Multicatalytic protease inhibitors

ABSTRACT

Disclosed herein are inhibitors of the multicatalytic protease enzyme which are represented by the general formula: ##STR1## Constituent members and preferred constituent members are disclosed herein. Methodologies for making and using the disclosed compounds are also set forth herein.

This application is a division of U.S. application Ser. No. 08/760,638filed Dec. 4, 1996, now U.S. Pat. No. 5,830,870, which is a division ofU.S. application Ser. No. 08/552,794 filed Nov. 3, 1995, now U.S. Pat.No. 5,614,649, which is a continuation-in-part of U.S. application Ser.No. 08/464,398 filed Jun. 5, 1995, abandoned, which is acontinuation-in-part of U.S. application Ser. No. 08/337,795 filed Nov.14, 1994, now U.S. Pat. No. 5,550,262.

FIELD OF THE INVENTION

This invention relates to inhibitors of multicatalytic protease (MCP),to compositions including such inhibitors and to methods for the use ofMCP inhibitors to, for example, retard loss of muscle mass incident tovarious physiological states.

BACKGROUND OF THE INVENTION

Eukaryotic cells constantly degrade and replace cellular protein. Thispermits the cell to selectively and rapidly remove proteins and peptideshasting abnormal conformations, to exert control over metabolic pathwaysby adjusting levels of regulatory peptides, and to provide amino acidsfor energy when necessary, as in starvation. See Goldberg, A. L. & St.John, A. C. Annu. Rev. Biochem. 45: 747-803 (1976). The cellularmechanisms of mammals allow for multiple pathways for protein breakdown.Some of these pathways appear to require energy input in the form ofadenosine triphosphate ("ATP"). See Goldberg, A. L. & St. John, supra.

Multicatalytic protease (MCP, also typically referred to as"multicatalytic proteinase," "proteasome," "multicatalytic proteinasecomplex," "multicatalytic endopeptidase complex," "20S proteasome" and"ingensin") is a large molecular weight (700 kD) eukaryoticnon-lysosomal proteinase complex which plays a role in at least twocellular pathways for the breakdown of protein to peptides and aminoacids. See Orlowski, M. Biochemistry 29(45) 10289-10297 (1990). Thecomplex has at least three different types of hydrolytic activities: (1)a trypsin-like activity wherein peptide bonds are cleaved at thecarboxyl side of basic amino acids; (2) a chirmotrypsin-like activitywherein peptide bonds are cleaved at the carboxyl side of hydrophobicamino acids; and (3) an activity wherein peptide bonds are cleaved atthe carboxyl side of glutamic acid. See Rivett, A. J. J. Biol. Chem.264: 21 12215-12219 (1989) and Orlowski, supra.

One route of protein hydrolysis which involves MCP also involves thepolypeptide "ubiquitin." Hershko, A. & Crechanovh, A. Annu. Rev.Biochem. 51: 335-364 (1982). This route, which requires MCP, ATP andubiquitin, appears responsible for the degradation of highly abnormalproteins, certain short-lived normal proteins and the bulk of proteinsin growing fibroblasts and maturing reticuloytes. See Driscoll, J. andGoldberg,, A. L. Proc. Nat. Acad. Sci. U.S.A. 86: 787-791 (1989).Proteins to be degraded by this pathway are covalently bound toubiquitin via their lysine amino groups in an ATP-dependent manner. Theubiquitin-conjugated proteins are then degraded to small peptides by anATP-dependent protease complex by the 26S proteasome, which contains MCPas its proteolytic core. Goldberg, A. L. & Rock, K. L. Nature 357:375-379 (1992).

A second route of protein degradation which requires MCP and ATP, butwhich does not require ubiquitin, has also been described. See Driscoll,J. & Goldberg, A. L., supra. In this process, MCP hydrolyzes proteins inan ATP-dependent manner. See Goldberg, A. L. & Rock, K. L., supra. Thisprocess has been observed in skeletal muscle. See Driscoll & Goldberg,supra. However, it has been suggested that in muscle, MCP functionssynergistically with another protease, multipain, thus resulting in anaccelerated breakdown of muscle protein. See Goldberg & Rock, supra.

It has been reported that MCP functions by a proteolytic mechanismwherein the active site nucleophile is the hydroxyl group of theN-terminal threonine residue. Thus, MCP is the first known example of athreonine protease. See Seemuller et al., Science (1995) 268 579-582;Goldberg, A. L, Science (1995) 268 522-523.

The relative activities of cellular protein synthetic and degradativepathways determine whether protein is accumulated or lost. The abnormalloss of protein mass is associated with several disease states such asmuscular dystrophy, cardiac cachexia, emphysema, leprosy, malnutrition,osteomalacia, child acute leukemia, and cancer cachexia. Loss of musclemass is also observed in aging, long term hospitalization or long termconfinement to bed, and in chronic lower back pain.

With denervation or disuse, skeletal muscles undergo rapid atrophy whichleads to a profound decrease in size, protein content and contractilestrength. This atrophy is an important component of many neuromusculardiseases in humans. Enhancement of protein breakdown has been implicatedas the primary cause of muscle wasting in denervation atrophy. Furono,K. et al. J. Biochem. 265/15: 8550-8557 (1990). While the specificprocess or processes involved in protein hydrolysis in muscle has notbeen identified, evidence is available linking the involvement of MCP inthe accelerated breakdown of muscle proteins. See, for example, Furono,supra., and PCT Published Application WO 92/20804 (publication date:Nov. 26, 1992).

MCP activity has been implicated in several disease states. For example,abnormally high expression of MCP in human leukemic cell lines has beenreported. Kumatori, A. et al. PNAS 87: 7071 (1990). Autoantibodiesagainst MCP in patients with systemic lupus erythematosus ("SLE") havealso been reported. Arribas, J. et al. J. Exp. Med. 173: 423-427 (1990).

Agents which are capable of inhibiting the MCP complex are needed; suchagents would provide a valuable tool for both those conducting researchin the area of, for example, MCP activity, as well as those in themedical fields in order to, for example, control the deleterious effectsof abnormal or aberrant MCP activity. The present invention is directedto these important ends.

SUMMARY OF THE INVENTION

The present invention is directed to novel multicatalytic protease("MCP") inhibitors. The subject invention also comprises methods forinhibition of MCP associated with certain disorders, including thetreatment of muscle wasting disorders.

In one aspect are provided compounds having formula: ##STR2##

Consitutent members are defined infra, as well as preferred constituentmembers.

The compounds of the invention are useful in a variety of applications.For example, the compounds may be employed in research applications tofurther refine and develop in vitro and in vivo models for mechanisticunderstanding of the MCP pathway and for presentation of peptideantigens via the major histocompatibility complex class I (MHC I)pathway.

In a clinical setting, compositions comprising the claimed compounds canbe used for inhibiting MCP activity, decreasing the loss of muscle mass,treating muscle wasting disorders, reducing superoxide dismutasedegradation and treating disorders characterized by a reduction ofsuperoxide dismutase activity.

Methodologies also are presented for making compounds of the invention.

These and other features of the compounds will be set forth in expandedform as the disclosure continues.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of embodiments of the disclosed MCP inhibitorson processing of electroporated OVA by M12.B6 cells.

FIG. 2 shows the effect of OVA concentration on the inhibition ofprocessing by an embodiment of the invention.

FIG. 3 shows the effect of OVA concentration on the inhibition ofprocessing by an embodiment of the invention.

FIG. 4 shows a physical map defining the SOD-1 gene and the location ofthe FALS mutations, restriction sites and the PCR primers.

FIG. 5 shows the quantitation of SOD-1 levels in transiently transfected293 cells after incubation with 5 μM of embodiments of the MCPinhibitors.

FIG. 6 shows the dose response of various SOD-1 isoforms to anembodiment of the invention.

FIG. 7 shows that SOD-1 turnover is a function of MCP activity.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

This invention provides MCP inhibitors, compositions including theseinhibitors and methods of using these inhibitors. The MCP inhibitors ofthe invention are represented, for example by the formula: ##STR3##wherein:

R₁ is selected from the group consisting of --C.tbd.N, --C(═O)OR₉,phthalimido, --NH--SO₂ R₉, and --NH--J;

R₂ is selected from the group consisting of H, hydroxyl, alkyl havingfrom one to ten carbons, and cycloalkyl having from three to sevencarbons;

R₃ is selected from the group consisting of --(CH₂)_(m)--NH--C(═N--R₅)--NH₂, --R₆ --NO₂, --R₆ --J, and --R₆ --CN;

R₄ is --CH(CH₂ --R₇)--Q;

Q is selected from the group consisting of --CH--R₈, --C(═O)CH₃,--C(═O)CH₂ Cl, --C(═O)CH₂ Br, --C(═O)CH₂ F, --C(═O)CHF₂, --C(═O)CF₃,--C(═O)C(═O)R₇, --C(═O)C(═O)NH--R₇, --C(═O)CO₂ --R₇, --C(═O)CO₂ H,--B(OH)₂, ##STR4## where p and q, independently, are 2 or 3; W iscycloalkyl;

R₅ is selected from the group consisting of --NO₂, --CN, and --J;

R₆ is --(CH₂)_(m) --NH--C(═NH)--NH--;

R₇ is selected from the group consisting of phenyl, and alkyl havingfrom one to eight carbons, said alkyl group being optionally substitutedwith one or more halogen atoms, aryl, or heteroaryl groups;

R₈ is selected from the group consisting of ═CO, ═N--NHC(═O)--NH₂,═N--OH, ═N--OCH₃, ═N--O--CH₂ --C₆ H₅, ═NNH--C(═S)--NH₂ and ═N--NH--J;

R₉ is s elected from the group consisting of hydrogen and alkyl havingfrom one to six carbons, said alkyl group being optionally substitutedwith one or more halogen atoms, aryl or heteroaryl groups;

J is a protecting group;

n is an integer from 3 to 10; and

m is an integer from 2 to 5.

In some preferred embodiments R₁ is --C.tbd.N, --C(═O)OCH₃, phthalimidoor --NH--SO₂ CF₃, and in other preferred embodiments R₂ is H orcyclopentyl.

R₃ is preferably --(CH₂)₃ --NH--C(═N--R₅)--NH₂.

Q is preferably --CH--R₈, --B(OH)₂, --C(═O)C(═O)NH--R₇, or has thestructure: ##STR5##

R₅ is preferably --NO₂, --CN, --PMC, --MTR, --MTS, or Tos.

R₇ is preferably --CH(CH₃)₂, --(CH₂)₂ --CH₃, --CH₂ --CH₃, or --C₆ H₅.

R₈ is preferably ═O, ═N--OH, ═N--O--CH₂ --CH₆ H₅, ═NNH--C(═O)--NH₂ or═NNH--C(═OS)--NH₂.

In some preferred embodiments R₁ is --C(═)OCH₃, phthalimido or --NH--SO₂CF₃ ; R₂ is cyclopentyl; R₃ is --(CH₂)₃ --NH--C(═N--NO₂)--NH₂ ; R₇ is--CH(CH₃)₂ ; and R₈ is ═O.

In other preferred embodiments R₁ is --C.tbd.N; R₂ is cyclopentyl; R₃ is--(CH₂)₃ --NH--C(═N--NO₂)--NH₂ or --(CH₂)₃ --NH--C(═N--J)--NH₂ ; R₇ is--CH(CH₃)₂ ; and R₈ is ═O.

In further preferred embodiments R₁ is C.tbd.N; R₂ is cyclopentyl; R₃ is--(CH₂)₃ --NH--C(═N--NO₂)--NH₂ or --(CH₂)₃ --NH--C(═N--J)--NH₂ ; R₇ is--CH(CH₃)₂ ; Q is --CH--R₈ ; and R₈ is ═N--NHC(═O)--NH₂, ═N--OH,═N--OCH₃, or ═N--O--CH₂ --C₆ H₅.

As used herein, the term "alkyl" is; meant to include straight-chain,branched and cyclic hydrocarbons such as ethyl, isopropyl andcyclopentyl groups. Substituted alkyl groups are alkyl groups for whichone or more hydrogen atoms have been replaced by halogen, otherhydrocarbon groups (for example, a phenyl group), a heteroaryl group, ora group in which one or more carbon atoms are interrupted by oxygenatoms. Preferred alkyl groups have 1 to about 8 carbon atoms. As usedherein, the term "halogen" has its usual meaning and includes fluorine,chlorine, bromine and iodine, with fluorine being a preferred halogen.The term "Arg" as used in the present invention has its normal meaningas the abbreviation for the amino acid "arginine."

In some embodiments compounds of thae invention contain protectinggroups. As used herein, the phrase "protecting groups" is to be accordeda broad interpretation. Protecting groups are known per se as chemicalfunctional groups that can be selectively appended to and removed fromfunctionalities, such as hydroxyl groups, amino groups and carboxylgroups. These groups are present in a chemical compound to render suchfunctionality inert to chemical reaction conditions to which thecompound is exposed. Any of a variety of protecting groups may beemployed with the present invention. One such protecting group is thephthalimido group. Other preferred protecting groups according to theinvention have the following formulas: ##STR6## Further representativeprotecting groups suitable for practice in the invention may be found inGreene, T. W. and Wuts, P. G. M., "Protective Groups in OrganicSynthesis" 2d. Ed., Wiley & Sons, 1991, the disclosures of which arehereby incorporated by reference in their entirety.

As previously indicated, MCP activity has been linked with a variety ofdisorders and diseases. Because compounds as disclosed herein are usefulin inhibiting the activity of MCP, and because the usefulness of suchcompounds can be applied to both research and therapeutic settings,methodologies for inhibiting the activity of MCP by contacting the MCPwith a compound of the invention include providing the compound to amammal, including a human, as a medicament or pharmaceutical agent.

As used herein, the term "contacting" means directly or indirectlycausing placement together of moieties to be contacted, such that themoieties come into physical contact with each other. Contacting thusincludes physical acts such as placing the moieties together in acontainer, or administering moieties to a patient. Thus, for example,administering a compound of the invention to a human patient evidencinga disease or disorder associated with abnormal and/or aberrantactivities of MCP which are associated with such disease or disorder,falls within the scope of the definition of term "contacting."

In preferred embodiments pharmaceutical compositions according to theinvention are administered to patients suffering from a disorder, i.e.,an abnormal physical condition, a disease or pathophysiologicalcondition associated with abnormal and/or aberrant activities of MCP.The disorders for which the compositions of the invention areadministered are preferably those which directly or indirectly produce awasting (i.e., loss) of muscle mass, that is, a muscle wasting disorder.These include muscular dystrophies, cardiac cachexia, emphysema,leprosy, malnutrition, osteomalacia, child acute lukemia, AIDS cachexiaand cancer cachexia.

In the context of the invention, "administering" means introduction ofthe pharmaceutical composition into a patient. Preferred methods ofadministration include intravenous, subcutaneous and intramuscularadministration. Preferably the compound will be administered as apharmaceutical composition comprising the compound in combination with apharmaceutically acceptable carrier, such as physiological saline. Othersuitable carriers can be found in Remington's Pharmaceutical Sciences(Mack Pub. Co., Easton, Pa., 1980).

The concentrations of the compounds described herein in a pharmaceuticalcomposition will vary depending upon a number of factors, including thedosage of the drug to be administered, the chemical characteristics(e.g., hydrophobicity) of the compounds employed, and the route ofadministration. In general terms, the compounds of this invention may beprovided in an aqueous physiological buffer solution containing about0.1 to 10% w/v compound for parenteral administration. Typical doseranges are from about 1 μg/kg to about 1 g/kg of body weight per day; apreferred dose range is from about 0.01 mg/kg to 100 mg/kg of bodyweight per day. The preferred dosage of drug to be administered islikely to depend on such variables as the type and extent of progressionof the disease or disorder, the overall health status of the particularpatient, the relative biological efficacy of the compound selected, andformulation of the compound excipient, and its route of administration.As used herein the term "patient" denotes any type of vertebrate.Preferably, the patient is a human.

The muscular dystrophies are genetic diseases which are characterized byprogressive weakness and degeneration of muscle fibers without evidenceof neural degeneration. In Duchenne muscular dystrophy (DMD) patientsdisplay an average of a 67% reduction in muscle mass, and in myotonicdystrophy, fractional muscle protein synthesis has been shown to bedecreased by an average of 28%, without any corresponding decrease innon-muscle protein synthesis (possibly due to impaired end-organresponse to anabolic hormones or substrates). Accelerated proteindegradation has been demonstrated in the muscles of DMD patients.

Severe congestive heart failure (CHF) is characterized by a "cardiaccachexia," i.e., a muscle protein wasting of both the cardiac andskeletal muscles, with an average 19% body weight decrease. The cardiaccachexia is caused by an increased rate of myofibrillar proteinbreakdown.

Emphysema is a chronic obstructive pulmonary disease, defined by anenlargement of the air spaces distal to the terminal non-respiratorybronchioles, accompanied by destructive changes of the alveolar walls.Clinical manifestations of reduced pulmonary functioning includecoughing, wheezing, recurrent respiratory infections, edema, andfunctional impairment and shortened life-span. The efflux of tyrosine isincreased by 47% in emphysematous patients. Also, whole body leacineflux remains normal, whole-body leucine oxidation is increased, andwhole-body protein synthesis is decreased. The result is a decrease inmuscle protein synthesis, accompanied by a decrease in whole bodyprotein turnover and skeletal muscle mass. This decrease becomesincreasingly evident with disease progression and long termdeterioration.

In diabetes mellitus, there is a generalized wasting of small muscle ofthe hands, which is due to chronic partial denervation (neuropathy).This is most evident and worsens with long term disease progression andseverity.

Leprosy is associated with a muscular wasting which occurs between themetacarpals of the thumb and index finger. Severe malnutrition ischaracterized by, inter alia, severe muscle wasting.

Osteomalacia is a nutritional disorder caused by a deficiency of vitaminD and calcium. It is referred to as "rickets" in children, and"osteomalacia" in adults. It is marked by a softening of the bones (dueto impaired mineralization, with excess accumulation of osteoid), pain,tenderness, muscle wasting and weakness, anorexia, and overall weightloss. It can result from malnutrition, repeated pregnancies andlactation (exhausting or depleting vitamin D and calcium stores), andvitamin D resistance.

In childhood acute leukemia there is protein energy malnutrition whichresults in skeletal muscle wasting. Studies have shown that somechildren exhibit the muscle wasting even before diagnosis of theleukemia, with an average 27% decrease in muscle mass.. There is also asimultaneous 33%-37% increase in adipose tissue, resulting in no netchange in relative boded weight and limb circumference.

Cancer cachexia is a complex syndrome which occurs with variableincidence in patients with solid tumors and hematological malignancies.Clinically, cancer cachexia is manifested as weight loss with massivedepletion of both adipose tissue and lean muscle mass, and is one causeof death which results from cancer. Cancer cachexia patients haveshorter survival times, and decreased response to chemotherapy. Inaddition to disorders which produce muscle wasting, other circumstancesand conditions appear to be linked in some fashion with a decrease inmuscle mass. Such afflictions include muscle wasting due to chronic backpain, advanced age, long term hospitalization due to illness or injury,alcoholism and corticosteroid therapy.

Studies have shown that in severe cases of chronic lower back pain,there is paraspinal muscle wasting. Decreasing paraspinal muscle wastingalleviates pain and improves function.

It is also believed that general weakness in old age is due to musclewasting. As the body ages, an increasing proportion of skeletal muscleis replaced by fibrous tissue. The result is a significant reduction inmuscle power, but only a marginal reduction in fat-free mass.

Studies have shown that in patients suffering injuries or chronicillnesses, and hospitalized for long periods of time, there islong-lasting unilateral muscle wasting, with an average 31% decrease inmuscle mass. Studies have also shown that this can be corrected withintensive physiotherapy. However, it may be more effective for manypatients to effect improvement with drug therapy.

In alcoholics there is wasting of the anterior tibial muscle. Thisproximal muscle damage is caused by neurogenic damage, namely, impairedglycolytic and phosphorylase enzyme activity. The damage becomesapparent and worsens the longer the duration of the alcohol abuse.Patients treated with corticosteroids experience loss of muscle mass.

MCP has been shown to activate the intracellular mediator ofinflammation referred to as NF_(kappa) B. See Baeuerle, P. A. andHenkel, T. (1994) Annu. Rev. Immunol. 12, 141-179. Inhibitors of MCPtherefore potentially have use in the treatment of autoimmune andinflammatory diseases.

The compounds of the invention can be used to alleviate the muscle massloss resulting from the foregoing conditions, as well as others.Additionally, the MCP inhibitors of the invention are useful inveterinary and animal husbandry applications to counter weight loss inanimals, or to promote growth.

MCP has been implicated in the presentation of peptide antigens via themajor histocompatibility complex class I (MHC I) pathway. See Goldbergand Rock, supra; see also Rock et al., Cell, 78: 761-771 (1994)hereinafter "Rock et al." Inhibitors of MCP therefore have utility asresearch reagents in studies where inhibition of the MCH I pathway isdesired as well as in the alleviation of diseases and disorders whichare associated with aberrant and/or abnormal MHC-I processing ofantigens. Because the precise origin of most of the peptides presentedon MHC-I molecules is still not clear and because evidence has recentlyaccumulated that MCP may play a role in MHC-I presentation (see Rock etal. supra), reagents such as the disclosed MCP inhibitors which blockthe proteolytic processing of antigens for MHC-I presentation would beuseful in resolving the importance of this pathway.

Surprisingly, it has also been found that MCP inhibitors of theinvention are also useful in enhancing the activity of Cu/Zn superoxidedismutase-1 ("SOD-1") enzyme. Accordingly, these compounds are useful inboth research settings for the investigation of SOD-1 deficient systemsand in the treatment of neurodegenerative or other disorderscharacterized by a reduction in SOD-1 enzyme activity (i.e., whereinsuch a reduction has been implicated in the pathogenesis of thedisorder). Such conditions include diseases involving oxidative stresssuch as Parkinson's disease, Alzheimers's disease, Huntington's disease,stroke, trauma, and ischemia.

SOD-1 is a homodimeric metalloenzyme that catalyzes the dismutation ofthe toxic superoxide anion O₂ to O₂ and H₂ O₂. SOD-1 is a scavenger offree radicals and therefore acts as a first line defense in thedetoxification of superoxide radicals, which are normal by-products ofaerobic metabolism. SOD-1 occurs primarily in eukaryotes and is found inthe cytoplasm of virtually all cell types. SOD-1 is an essential enzymein the physiological response to oxygen toxicity and has been activelyinvestigated as a therapeutic agent in pathological conditions relatedto oxidative stress. See Bannister et al., CRC Crit. Rev. Biochem. 22:111-180 (1987); Halliwell et al., Methods in Enzymol., 186: 1-75 (1990);Greenwald, Free Rad. Biol. Med. 8: 201-209 (1990).

Features that have prevented the use of SOD-1 as a therapeutic agent areits poor intracellular access when supplied exogenously, and itsextremely short half-life in serum. Therefore, compounds that enhancethe activity of intracellular SOD-1 would provide a significantadvancement in SOD-1 therapy.

ALS is a progressive paralytic disorder caused by degeneration of largemotor neurons of the spinal cord and brain. Approximately 5-10% of ALScases are familial (FALS) and are inherited as an autosomal dominanttrait. Recently, sixteen different missense mutations have beenidentified in a subset of families with FALS and occur within the geneencoding SOD-1. See Rosen, D. R., et al., Science 261:1047-1051 (1993);Deng, H.-X., et al., Nature 362:59-62 (1993). These mutations lead to adecrease in SOD-1 activity in red blood cells and brain tissue, and havebeen shown to destabilize the SOD-1 protein resulting in increasedturnover of the enzyme. See Bowling, A. C., et al., J. Neurochem. 61:2322-2325 (1993); Borchelt, D. R., et al., Proc. Natl. Acad. Sci. 91:8292-8296 (1994). Additionally, a transgenic-mouse model of ALS, basedupon the implication of the connection between SDD-1 and ALS, has beendescribed. Brown, R. H. 331/16 NEJM 1901 (1994).

We have discovered that our MCP inhibitors are potent positive effectorsof SOD-1. A preferred MCP inhibitor, referred to herein as "Compound14," specifically reduces wild-type and mutant SOD-1 degradation in adose-dependent manner (compound number designations are based upon theExample number which discloses the synthesis of the compound, e.g., thesynthesis of Compound 14 is set forth in Example 14, infra). As usedherein, reduction of SOD-1 degradation means retarding the rate at whichthe SOD-1 protein is catabolized.

The invention is further illustrated by way of the following examples.These examples are intended to further elucidate the invention, and arenot intended to limit the scope of the appended claims.

EXAMPLE 1

Isolation of multicatalytic protease from human tissues

Samples of human liver and brain obtained post-mortem were used forisolation and partial purification of MCP by ion-exchangechromatography, ammonium sulfate precipitation, and gel filtration(e.g., Driscoll and Goldberg, Proc. Natl. Acad. Sci. 86, 787-791 (1989);Yamamoto et al., Biochim, Biophys. Acta 882, 297-304 (1986). For eitherstarting material, tissue was homogenized in 10 volumes of 20 mMTris-HCl (pH 7.5) containing 20% glycerol. Following centrifugation at40,000×g for 30 minutes, proteolytic activity of the chymotrypsin-likecomponent of MCP could be detected in the supernatant (see below). Thesupernatant was fractionated on a DEAE-Sepharose Fast Flow columnequilibrated in homogenization buffer. For each liter of supernatant,250 ml of resin was used. Following sample loading, the column waswashed with .sup.˜ 10 volumes homogenization buffer, and proteins wereeluted with a linear NaCl gradient of from 0 to 400 mM (21 for 250 mlresin). Fractions were assayed for MCP activity, and the activefractions were pooled and subjected to precipitation with (NH₄)₂ SO₄ at80% saturation. Precipitated proteins were collected by centrifugation,resuspended in homogenization buffer, and loaded on a Sephacryl S300HRcolumn (500 ml resin volume) that had been standardized using bovineserum albumin (68 kDa). A single peak of MCP activity was eluted with amolecular weight of .sup.˜ 650 kDa. This preparation was free of othermeasurable proteolytic activities and maintained its MCP activity uponstorage at 4° C. for >6 months. This preparation was used for most ofthe experiments. Further fractionation of the preparation on anhydroxyapatite-Ultrogel column (Yamamoto et al., supra) yielded a morehighly purified enzyme, which according to denaturing SDS-polyacrylamidegel electrophoresis was comprised of the expected >10 subunits rangingin M_(r) from 20 to 35 kDa.

EXAMPLE 2

Assay for chymotrypsin-like and trypsin-like activities of MCP

The procedure for MPC isolation described above generated an enzymecomplex whose proteolytic activities were latent, but which could beactivated by addition of low concentrations (0.02-0.05%) of SDS(Yamamoto et al., supra). The chymotrypsin-like activity was assayedaccording to the following procedure: in 96 well microtiter plates,human MCP was diluted 4 to 10-fold in homogenization buffer containing0.04% SDS. A calorimetric substrate MeOSuc-EVKM-para-nitroanilide(methoxysuccinyl-Glu-Val-Lys-Met-pNa), purchased from Bachem BioscienceInc., King of Prussia, Pennsylvania was added to a final concentrationof 100 μM from a stock solution of 10 mM in dimethylsulfoxide. Reactionvolumes were 200 μl per well. After incubation for various periods oftime at 37° C., the concentration of free pNa was determined on aBiotech EL-340 microplate reader, reading absorption at 490 nm. Proteaseactivity was determined under conditions in which substrate hydrolysisincreased linearly with time and the change in absorption wasproportional to the concentration of free pNa. Alternatively, afluorogenic substrate was used,methoxysuccinyl-Phe-Leu-Phe-amidomethylcoumarin (Enzyme SystemsProducts, Dublin, Calif.), and the change in fluorescence monitored atan excitation of 390 nm, and an emission at 460 nm.

The trypsin-like activity of human MCP was assayed as described abovewith the following modifications. Reactions were carried out inTris-glycerol buffer (pH 9.5) supplemented with 1 mM 2-mercaptoethanol,and the substrate was a fluorogenic substrate,Benzyloxycarbonyl--Phe--Arg--AMC (100 μM).

After incubation for various periods of time at 37° C., theconcentration of free AMC was determined on a Fluoroskan IIspectrofluorimeter with an excitation filter of 390 nm and an emissionfilter of 460 nm. Protease activity was determined under conditions inwhich substrate hydrolysis increased linearly with time and the changein fluorescence was proportional to the concentration of free AMC.

EXAMPLE 3

Determination of IC₅₀ values for MCP Inhibitors

IC₅₀ values are typically defined as the concentration of a compound (inthis case, the disclosed MCP inhibitor) necessary to produce 50%inhibition of the enzyme's activity. IC₅₀ values are useful indicatorsof the activity of a compound for its designated use. Preferably, theinhibitors of the invention have IC₅₀ values of less than about 10micromolar.

Inhibition of the chymotrypsin-like or trypsin-like activity of MCP wasdetermined by incubating the enzyme with various concentrations ofputative inhibitors for 15 minutes at 37° C. prior to the addition ofsubstrate. Each experimental condition was evaluated in triplicate, andreplicate experiments were performed for the inhibitors describedherein.

EXAMPLE 4

Demonstration of Inhibition of Cellular Muscle Breakdown: Inhibition ofUnweighting Atrophy in Juvenile Rats

The effect of several inhibitors on the unweighting atrophy of thesoleus muscle in juvenile rats was determined. See Tischler, M. E.(1990) Metabolism 39/7: 756-763 (hereinafter "Tischler--1990") for ageneral discussion of the procedure.

Juvenile female Sprague-Dawley rates (80-90 g) were tail-cast, hind limbsuspended as in Jetspers, S. R. and Tischler, M. E., (1984) J. Appl.Physiol. 57: 1472-1479. The animal's hind limbs were elevated above thefloor of the cage with each animal housed individually. Animals had freeaccess to food and water, and were weighed at the time of suspension andat time of termination. During the suspension period the animals werechecked daily to ensure that their toes were not touching the floor ofthe cage, and that there was no swelling of the tail due to the cast.

A. Experimental Design--Part 1

Each experiment began with the suspension of 20 rats which were randomlydivided into 4 groups of 5 animals each. Group A was suspended for just2 days and provided baseline data to approximate the soleus muscle sizein other animals suspended for longer times. Average body weights forthe groups at the outset of the study were compared and used as acorrection factor for body size differences. Group B was a secondcontrol group which had the soleus of one limb treated with an aqueoussolution of mersalyl after two days of unweighting, to demonstrate theability to slow muscle atrophy during unweighting, for each group ofanimals. Mersalyl has been previously studied and demonstrated toprevent atrophy in an in vivo model substantially as described in theprotocol utilized herein. See Tischler--1990. At 2 days afterunweighting commenced, an aqueous solution of mersalyl (200 nM; 4 μl/100g initial body wt) was injected into one soleus, as describedpreviously. The contralateral muscle was injected with a similar volumeof 0.9% saline ("Vehicle"). The animals were maintained underInnovar-vet (10 μl/100 g body wt) tranquilization during the in situinjection procedure. After the injections, the animals were suspendedfor an additional 24 hours and the soleus was removed. Groups C and Dfor each experiment were used for testing each of two differentembodiments of the disclosed compounds.

Animals were treated as in group B, except that 1 mM MCP inhibitor,contained in dimethysulfoxide (DMSO), was injected into the soleus ofone leg and DMSO only into the contralateral soleus. Thus eachexperiment consisted of two control groups and the testing of MCPinhibitors of the invention. The completion of five such experimentswith different pairs of inhibitors provided for an "n" value of 10 fortesting each MCP inhibitor and with each tested in two differentshipments of animals.

B. Processing of the Soleus Muscle--Part 1

After the animal was sacrificed, the soleus was excised, trimmed of fatand connective tissue, and carefully weighed. The muscle was thenhomogenized in 10% trichloroacetic acid (TCA) and the precipitatedprotein pelleted by centrifugation. The pellet was then washed once with10% TCA and once with ethanol:ether (1:1). The final pellet wassolubilized in 4 ml of 1N sodium hydroxide. The sample was then analyzedfor protein content by the biuret procedure, using albumin as astandard.

C. Data Analysis--Part 1

The effect of MCP inhibitors on total muscle protein content wasexamined primarily by paired comparison with the untreated contralateralmuscle. The ratio of contents was calculated and then analyzedstatistically by analysis of variance ("ANOVA"). The left leg was alwaysthe treated leg so that the protein content ratios could be compared tothe non-treated control animals as well. In this way, a significantdifference can be shown by comparing the protein content of the twolegs, as well as the relative effectiveness of the tested MCPinhibitors. A paired student test was also performed for the effect ofeach separate treatment. The non-treated control data also provided anestimate of protein content of day 2. This allowed approximation of theprotein changes over the 24 hours of treatment for each of the Groups B,C, and D.

D. Experimental Design--Part 2

Each experiment consisted of 10 animals with groups of 5 animals beingtested with one of the inhibitors for its effect on protein synthesis.Control animals were not needed for this aspect of the study as thecontralateral DMSO-treated muscle served as the paired control for theinhibitor-treated muscle. Each group was injected as described forgroups C and D in part 1. Twenty-four hours after the in situ treatmentthe fractional rate of protein synthesis was analyzed in both soleusmuscles. Each muscle was injected with a 0.9% saline solution (3.5μl/100 g final body wt) containing ³ H-phenylalanine (50 mM; 1 μCi/μl).Fifteen minutes later the middle two-thirds of the muscle was excisedand the muscle was processed as described below.

E. Processing of the Soleus Muscle--Part 2

The muscle was first washed for 10 minutes in 0.84% saline containing0.5 mM cycloheximide, to terminate protein synthesis, and 20 mMcycloleucine, to trap phenylalanine in the cell. The muscle was thenhomogenized in 2.5 ml of ice-cold 2% perchloric acid. The precipitatedprotein was pelleted by centrifugation. One aliquot of the supernatantwas taken for liquid scintillation counting and another aliquot wasprocessed for conversion of phenylalanine to phenethylamine to determinethe soluble phenylalanine concentration fluorometrically. See Garlick,P. J. et al,, Biochem. J., 192 719-723 (1980) and Munoz, K. M. et al.,.1993 Meatbolism, in press. These values provide the intracellularspecific activity. The specific activity of phenylalanine in the muscleprotein was determined after hydrolyzing the protein by heating in 6NHCl. The amino acids released were solubilized in buffer. One aliquotwas taken for scintillation counting and another for analysis ofphenylalanine as for the supernatant fraction. The fractional rate ofprotein synthesis was calculated as: protein specificactivity/intracellular specific activity x time.

F. Data Analysis--Part 2

Analyses of protein synthesis were on a paired basis for each MCPinhibitor. Student paired t test comparisons of the contralateralmuscles determined whether there was any effect of the inhibitor onprotein synthesis. Protein breakdown can be calculated approximately asthe fractional rate of protein synthesis (from part 2) plus thefractional rate of protein accretion (from part 1), where protein lossyields a negative value for protein accretion.

Qualitatively the ability of MCP inhibitors to slow protein loss withoutaffecting protein synthesis indicates a slowing of protein degradation.

G. Results

Table 1 shows the lack of effect of the MCP inhibitors on controlmuscle.

Table 2 shows the change in protein content during the third day ofunweighting.

Table 3 shows the effect of the MCP inhibitors on unweighted muscleprotein.

Table 4 shows the effect of the MCP inhibitors on unweighted musclesynthesis. In the Tables, the compound number refers to the Examplenumber where the synthesis route for the compound is set forth.

                  TABLE 1                                                         ______________________________________                                        Lack of Effect of MCP Inhibitors on Control Muscle                                          Protein (mg/muscle)                                             Treatment       Vehicle Treatment                                             ______________________________________                                        DMSO            5.9     6.7                                                   Compound 34     6.1     6.3                                                   Compound 14     6.0     6.1                                                   Compound 20     5.7     5.9                                                   ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Change in Protein Content During Day 3 of Unweighting                                   Protein (mg/muscle)                                                                            Effect                                             Treatment   Start    End       (%)   P                                        ______________________________________                                        Saline      6.5      5.7       -12   <0.001                                   DMSO        6.5      5.9       -10   <0.001                                   Mersalyl    6.4      6.6       +3    <0.05                                    Compound 34 6.3      6.6       +5    <0.05                                    Compound 14 6.8      6.4       0     >0.1                                     Compound 20 6.5      6.2       -4    <0.07*                                   Compound 16 6.3      6.0       -5    <0.07*                                   ______________________________________                                         *marginally significant values                                           

                  TABLE 3                                                         ______________________________________                                        Effect of MCP Inhibitors on Unweighted Muscle Protein                                   Protein (mg/muscle)                                                                            Effect                                             Treatment   Vehicle Treatment  (%)   P                                        ______________________________________                                        Mersalyl    5.7     6.6        14    <0.001                                   Compound 34 5.9     6.6        12    <0.001                                   Compound 14 6.0     6.4        7     <0.06*                                   Compound 20 5.9     6.2        5     <0.95                                    Compound 16 5.9     6.0        0     >0.1                                     ______________________________________                                         *marginally significant values                                           

                  TABLE 4                                                         ______________________________________                                        Lack of Effect of MCP Inhibitors on                                           Unweighted Muscle Synthesis                                                                 Protein (mg/muscle)                                             Treatment       Vehicle MCP Inhibitor                                         ______________________________________                                        Compound 34     14.5    13.3                                                  Compound 14     13.1    13.8                                                  Compound 20     14.1    14.6                                                  ______________________________________                                    

EXAMPLE 5

Demonstration of MCP Inhibition Using MHC-1 Processing

Compounds of the invention were assayed for the ability to inhibit theprocessing of exogenous OVA antigen by the class I majorhistocompatibility pathway (MHC-I). The MCP inhibitors were applied to afunctional antigen processing system that allows inclusion of theinhibitor within the antigen processing cell during exposure to antigen,followed by fixation of the processing cell. T cell hybridomas are thenadded to the processing cells in the absence of inhibitor to determinethe expression of peptide-MHC-I complexes, which reflects antigenprocessing prior to fixation.

A. Cell Culture

Cells were grown at 37° C. in a humidified atmosphere maintained at 5%CO₂ in a standard medium: DMEM (Gibco, St. Louis, Mo.) with 10% fetalcalf serum (Hyclone, Logan Utah), 5×10⁻⁵ M 2-mercaptoethanol,antibiotics and the following supplements: L-arginine HCl (116 mg/L),L-asparagine (36 mg/L), NaHCO₃ (2 g/L), sodium pyruvate (1 mM). See alsoHarding, C. V., (1992) Eur. J. Immunol. 22: 1865-1869. M12.B6 cells(generous gift of Osami Kanagawa, Washington University, ST. Louis, Mo.)were used for antigen presentation; they were created by fusing M12.C3murine B lymphoma cells with LPS-stimulated splenocytes (source of Blymphoblasts) from a C57BL/6 mouse. Accordingly, they express theH-2^(b) antigens. DOWB T hybridoma cells are specific for the OVA(323-339) peptide bound to either I-A^(b) or I-A^(d). See Yewdell etal., Science 1988 239: 637. B3.1 T hybridoma cells respond to OVA(258-276)-K^(b).

B. Electroporation and antigen processing studies

To be processed and presented by the MHC-1 pathway, the exogenousantigen (ovalbumin; OVA) must penetrate into the cytosol of theprocessing cells. OVA was introduced into the cytosol of processingcells (M12.B6 cells) by means of electroporation.

Electroporation was performed with a Gibco BEL Electroporator using 0.4cm gap cuvette chambers. Capacitance was 800 μF and voltage was 50-300V. Electroporation was performed in serum-free RPMI or PMEM (Gibco) with1.5×10⁷ M12.B6 cells/ml (0.5×10⁷ -4.0×10⁷ range) in the presence of OVAat 4° C. The cells were immediately placed on ice. Various inhibitorswere then added and the cells were transferred to 18° C. or 37° C.Finally the cells were lightly fixed for about 10 minutes with 1%paraformaldehyde and washed extensively at 37° C., preventing furtherprocessing. The extent of processing, (i.e., the level of specificpeptide-MHC complexes expressed) was determined by the ability of theM12.B6 cells to stimulate IL-2 secretion by B3.1 or DOBW T hybridomacells. T cells (10⁵) were plated with the M12.B6 cells (2×105 if fixed,5×10⁴ if viable) in 0.2 ml for 18-24 hours at 37° C. in a 10² chamber.Both T hybridomas respond to antigen stimulation by the secretion ofIL-2 (assayed in the supernatants by IL-2-dependent CTLL cellproliferation and [H³ ] thymidine incorporation. See Allen et al., J.Imunol. 132: 1077).

Results of these studies are shown in FIGS. 1-3. FIG. 1 shows theeffects of Compounds 14 and 16 on the processing of electroporated OVAby M12.B6 cells. FIGS. 2 and 3 show the effect of Compounds 14 and 20 onMHC-1 OVA processing. Both compounds effectively inhibit OVA processing.These data provide additional support for MCP inhibitory effect of thecompounds of the invention. It is accepted that the classical MHC-Ipathway involves antigen degradation by proteasomes. See Goldberg etal., supra. Thus, the compounds can be utilized in further understandingthe importance of the proteolytic processing of antigens.

EXAMPLE 6

A. Reduction of Cu/Zn Superoxide Dismutase (SOD-1) Degradation

I. Part 1--Cloning and mutagenesis.

A mouse SOD-1 cDNA clone was obtained from Jim Mahaffey (North CarolinaState University, Raleigh, N.C.). This clone was modified by polymerasechain reaction (PCR) methodologies to incorporate 1) a Kozak translationinitiation consensus signal (i.e., 5'-GCCGCCACC-3') directly upstream ofthe ATG start codon, 2) a Hind III restriction site 5' of this consensussignal and 3) an Xho I restriction site at the 3' terminus of the cDNA.The oligonucleotide primers used for the PCR procedures were: 5' primer(EH87)=5'-TCGATCGAAGCTTGCCGCCACCATGGCGATGAAAGC-3' (SEQ ID NO:1), 3'primer (EH88)=5'-AGCTAGCCTCGAGCAGATTACAGTTTAATG-3' (SEQ ID NO:2). Theresulting PCR product was digested with the restriction enzymes Hind IIIand Xho I and cloned into the Hind III+Xho I digested vector pBluescriptII SK+ (Stratagene Cloning Systems, La Jolla, Calif.) to yield pSK-HX2.

The FALS point mutations at amino acid 4 (Ala⁴ →Val) (GCG→GTG) and aminoacid 113 (Ile¹¹³ →Thr) (ATT→ACT) were introduced into the SOD-1 cDNAclone pSK-HX2 using the overlap extension PCR mutagenesis proceduredescribed by Ho et al., Gene 77: 51-59 (1989). The Ile¹¹³ →Thr mutantwas constructed as follows: the 5' PCR fragment was generated using the5' primer E.H78; consisting of the nucleotide sequence5'-TTAATCCTCACTCTAAGAAAC-3' (SEQ ID NO:3) (nucleotides 193 to 213 ofSOD1 cDNA where #1 is the A of the ATG; start codon) and the 3' primerEH84; consisting of the nucleotide sequence5'-TTGTACGGCCAGTGATGGAATGCT-3' (SEQ ID NO:4) (nucleotides 351 to 328),the 3' PCR fragment was constructed using the 5' primer EH83; consistingof the nucleotide sequence 5'-AGCATTCCATCAfTGGCCGTACAA-3' (SEQ ID NO:5)(nucleotides 328 to 351) and the 3' primer EH42; consisting of thenucleotide sequence 5'-TAATACGACTCACTATAGGG-3' (SEQ ID NO:6)(nucleotides 626 to 645 of pBluescript II SK+). Fifty nanograms of both5' and 3' PCR fragments were combined in the second step PCR reactionand amplified using primers EH78 and EH42. This PCR product was digestedwith Bal I and Xho I and cloned into Bal I+Xho I digested pSK-HX2 thusreplacing the native 255 bp Bal I/Xho I fragment with the FALS mutantfragment (clone pSK-113).

The Ala⁴ →Val mutant was constructed as follows: the 5' PCR fragment wasgenerated using the 5' primer EH105; consisting of the nucleotidesequence 5'-GCACACCACTTTCATCGCCATGGTGGCGGCAAGCTTCGATC-3' (SEQ ID NO:7)(nucleotides +21 to -20) and the 3' primer EH41; consisting of thenucleotide sequence 5'-ATTAACCCTCACTAAAGGGA-3' (SEQ ID NO:8)(nucleotides 792 to 773 of pBluescript II SK+). The 3' PCR fragment wasgenerated using the 5' primer EH104, consisting of the nucleotidesequence 5'-GATCGAAGCTTGCCGCCACCATGGCGATGAAAGTGGTGTGC-3' (SEQ ID NO:9)(nucleotides -20 to +21) and the 3' primer EH103; consisting of thenucleotide sequence 5'-CTTCAGTTAATCCTGTAA-3' (SEQ ID NO:10) (nucleotides123 to 106). As above, 50 ng of both 5' and 3' PCR fragments werecombined in the second step PCR reaction and amplified using primersEH41 and EH103. This PCR product was digested with Hind III and Rsr IIand cloned into Hind III+Rsr II digested pSK-HX2 thus replacing thenative 51 bp Hind III/Rsr II fragment with the FALS matant fragment(clone pSK-A4V). All PCR products describes above were sequenced usingthe Sequenase method (US Biochemical, Cleveland, Ohio) to confirm thepresence of the mutations and the absence of any PCR errors. Anillustration of the SOD-1 gene defining the locations of the mutations,primers and restriction sites is shown in FIG. 4.

The SOD-1 cDNA expression vectors were constructed by digesting thewild-type (pSK-HX2) and mutant constructs (pSK-113 and pSK-A4V) withHind III and Xho I. Each of the three resulting 546 bp fragmentscontaining the SOD-1 CDNA sequences were separately cloned into HindIII+Xho I digested pcDNA I/Neo (Invitrogen Corp., San Diego, Calif.) toyield, respectively, pCMV-HX5 (wild-type SOD), pCMV-113 (Ile¹¹³ →Thr),and pCMV-A4V (Ala⁴ →Val).

II. Part 2.

Initial experiments were conducted to determine whether the reducedstability of the FALS mutant SOD-1 proteins was due to MCP-mediatedproteolytic degradation. The human kidney cell line designated 293(human 293 cells or 293 cells)was obtained from the American TypeCulture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md., 20852(ATCC #CRL 1573) and grown in Dulbecco's modified Eagle's medium with4.6 g/L glucose, 10% heat-inactivated horse serum (Gibco/LifeTechnologies Inc. Gaithersburg, Md.). The human 293 cells weremaintained at 37° C. in an atmosphere of 5% CO₂. The 293 cells weretransiently transfected using the calcium phosphate method (Chen, C. etal., Biotechniques 6: 632 (1988). The pCMV-113 SOD-1 expression vectorwas introduced into 293 cells and 24 hours later, the cells wereincubated with either compound 34, compound 14, or compound 24, all at aconcentration of 5 μM, for a duration of 24 hours. The known MCP andother protease inhibitory compounds were solubilized in dimethylsulfoxide (DMSO). Other 293 cell cultures were incubated with an equalvolume of DMSO or left in medium only as negative controls.

Approximately 48 hours prior to receiving test compounds, 5×10⁵ 293cells were seeded in 36 mm dishes and maintained at 37° C. in anatmosphere of 5% CO₂. Cells were incubated with MCP inhibitors (compound34, 14 or 24) or with DMSO as a negative control for a duration of 24hours. Cell lysates were prepared by lysing the cells in approximately75 μl of phosphate-buffered saline (PBS) by freeze/thaw cycling. Proteinconcentrations of the cell lysates were determined using the BCA method(Pierce, Rockford, Ill.) and 2 to 2.5 μg of each sample waselectrophoresed on a 4-20% polyacrylamide geol (Novex, San Diego,Calif.) using a Tris/glycine/SDS (25 mM Tris/192 mM glycine/0.1% SDS)buffer system. Proteins were transferred to nitrocellulose filters byelectroelution and filters were blocked by incubation in blotto solution(5% dry milk in 25 mM Tris-buffered saline (1× TBS)) for 30 minutes.Filters were transferred to primary antibody solution (1:10,000 dilutionin blotto solution) and incubated for 2-18 hours. The primary antibodyused in these studies was polyclonal rabbit antiserum raised againstpurified mouse SOD-1 produced in E. coli (Hazelton Research Products,Denver, Pa.). The filters were washed three times for 5 minutes each in1×0 TBS and incubated in secondary antibody solution (1:2,000 dilutionin blotto solution) for two hours. The secondary antibody was a goatanti-rabbit IgG conjugated to alkaline phosphatase (Bio-Rad, Richmond,Calif.). Filters were washed three times for 5 minutes each in 1× TBSand stained for alkaline phosphatase activity by incubation for 5-60minutes in a commercially available alkaline phosphatase detectionreagent (Bio-Rad, Richmond, Calif.). Stained bands corresponding toSOD-1 protein were quantitated using a DocuGel V image analysis systemand RFLPscan software (Scanalytics, Billerica, Mass.). Levels of SOD-1are expressed as units of induction relative to the negative control(i.e., units of experimental divided by units of control). Immunoblotanalysis of cell lysates revealed that SOD-1 levels were modestlyelevated compared to those of controls in each of the three samplesincubated in the presence of MCP inhibitors (FIG. 5). These results showthat inhibition of the MCP by the compounds of the invention leads toincreased accumulation of SOD-1 protein in the Ile¹¹³ →Thr mutant andthereby implicates MCP as being responsible for the reduced levels ofSOD-1 in the cells containing the FALS mutation.

III. Part 3.

To further show that MCP activity is responsible for SOD-1 turnover,studies were conducted on cell lines stably producing wild-type mouseSOD-1 or the FALS mutant proteins, i.e., Val in place of Ala⁴ and Thr inplace of Ile¹¹³. Cell lines stably expressing mouse native SOD-1 and thetwo FALS mutant SOD-1 proteins mentioned above were derived by calciumphosphate transfection (Chen, C. et al., Biotechniques 6: 632 (1988)) of293 cells with SOD-1 cDNA expression constructs (described above)followed by selection for neomycin resistance by growth in the presenceof 1 mg/ml G418 (Geneticin™, Gibco/Life Technologies, Inc.,Gaithersburg, Md.). Cells were maintained in G418 selection for threeweeks at which time drug selection was removed and the G418-resistantcolonies of each transformed cell type were pooled for further growth.Compound 14 was utilized in these studies.

Cell lines expressing the indicated SOD-1 isoforms were incubated withvarious concentrations of compound 14 for 24 hours at which time theSOD-1 levels were measured by densitometric scanning of immunoblots.Levels of SOD-1 are expressed as units of induction relative to thesamples from negative controls (as above) and each data point is theaverage of three experiments. Dose response analysis of Compound 14indicated that incubation of the transformed cells with this MCPinhibitor led to significant accumulations of SOD-1 protein levels withmaximal accumulations occurring in those cells incubated with Compound14 at a concentration of approximately 20 μM (FIG. 6). Moreover, themagnitude of SOD-1 accumulation correlates with the estimated half-livesof the various SOD proteins; wild-type SOD-1 being the most stable andAla⁴→ Val the least stable (Borchelt D. R. et al., Proc. Natl. Acad.Sci. 91: 8292-8296 (1994)). These data are consistent with thehypothesis that the FALS mutations destabilize the SOD-1 proteins,possibly due to misfolding, and as a result of structural alterationsresulting from the mutations target these proteins for degradation byMCP. These results also demonstrate that Compound 14 has a statisticallysignificant effect on turnover of the wild-type SOD-1 protein furthersupporting the role of the MCP in SOD-1 degradation.

IV. Part 4.

To demonstrate the specificity of MCP activity in SOD-1 turnover (andrule out the possible involvement of other major proteolytic pathwayssuch as the calcium-activated protease calpain and the lysosomal.proteases), experiments were performed in which two transformed celllines (described above, one producing wild-type protein and the otherSOD-1 protein, with the Ala⁴ →Val mutation), were incubated with variousprotease inhibitors each of which is specific for a unique proteolyticactivity: Compound 14 inhibits MCP; "Calpeptin" (Novabiochem USA, LaJolla, Calif., cat no.03-34-0051; CBZ-Leu-Nle-aldehyde, Biochem.Biophys. Res. Comm. (1988) 153: 1201.), a cell penetrating "calpain"inhibitor (calpain is a cysteine protease); and DK-3 (Enzyme SystemsProducts, Dublin, Calif. CBZ-Phe-Ala-CHN₂) inhibits lysosomal proteaseactivity. In addition, cells were incubated with Compound 16 which is aless potent MCP inhibitor compared to a particularly preferred compoundof the invention, i.e. Compound 14. Cell lines expressing the indicatedSOD-1 isoforms were incubated for 24 hours with the following proteaseinhibitors: Compound 14, 20 μM; Calpeptin, 10 μM; DK-3, 5 μM; Compound16, 20 μM; or with DMSO as a negative control. After the 24 hours ofincubation with the inhibitors, SOD-1 levels were measured bydensitometric scanning of immunoblots. Levels of SOD-1 are expressed asunits of induction relative to untreated samples, as above. Each datapoint is the average of results from three experiments.

Results are presented in FIG. 7. It can be seen that the turnover ofAla⁴ →Val mutant SOD-1 is a function of MCP activity and only thespecific inhibition of MCP by the preferred Compound 14 leads to asignificant (three-fold) increase in the accumulation of SOD-1 protein.Incubation with the inhibitors of calpain or lysosomal proteases had noappreciable effect upon SOD-1 turnover. Further, Compound 14 treatmentresulted in a slight increase in wild-type SOD-1 accumulation, a findingalso observed previously in the dose response studies. Taken together,these studies demonstrate that MCP activity is critical for SOD-1turnover in the transformed 293 cell lines, and that inhibition of MCPby Compound 14 leads to an elevation of SOD-1 protein within the cell.

Synthesis of Inhibitors

The following provides exemplary synthesis routes for the preparation ofcompounds of the present invention. Other synthesis protocols, as wellas modifications of the following synthesis schemes will be readilyapparent to those skilled in the art once armed with thee presentdisclosure.

The inhibitors of the invention incorporate amide bonds which may beintroduced by well-known synthetic procedures using the solution-phasetechniques described in The Peptides: Analysis, Synthesis, Biology,Volume I (1979), eds. Gross, et al, Academic Press and described indetail in Examples 8-11 below.

The inhibitors may be prepared by the separate synthesis and coupling ofthe fragments:

    R.sub.1 --(CH.sub.2).sub.n --CH(R.sub.2)--COOH

and

    H.sub.2 N--CH(R.sub.3)--CONH--R.sub.4

(Coupling Procedure I) as described in Examples 14-38.

Alternatively, they may be prepared by the separate synthesis andcoupling of the fragments:

    R.sub.1 --(CH.sub.2).sub.n --CH (R.sub.2)--CONH--CH (R.sub.3)--COOH

and

    H.sub.2 N--R.sub.4

(Coupling Procedure II) as described in Examples 39-42 below.

When substituent Q contains an aldehyde group (i.e. when H₂ N--R₄ is anaminoaldehyde derived from an aminoacid, e.g. H₂ N--CH(CH₂ R₇)--CHO) therequisite acetal-protected aminoaldehyde may be synthesized as describedby Gacek, et al. Tetrahedron, 30, 4233 (1974) or by the proceduresdescribed in Example 12. After the coupling is completed (via CouplingProcedures I or II) the acetal protecting groups may be removed asdescribed in Example 11 (Method E) to liberate the free aldehyde group.

When Q is a methylketone or a diazo-, bromo-, or chloromethyl ketone(i.e. when H₂ N-R₄ is an alpha-amino-substituted methyl ketone or analpha diazo-, bromo- or chloromethyl ketone derived from an amino acid),Coupling Procedure II is particularly convenient. The hydrochloride saltof the chloromethyl ketone may be purchased (Bachem Bioscience Inc.,King of Prussia, Pa.) or can be prepared by conversion of theBoc-protected amino acid to a mixed carbonic anhydride followed byreaction with diazomethane to give the diazomethyl ketone. The bromo-and chloro- methyl ketones are prepared by reaction of the diazomethylketone with hydrogen bromide or chloride as described by Kettner, et al.Arch. Biochem. Biophys. 162, 56(1974), Fittkau, J. Prakt. Chem. 315,1037 (1973) or Zimmerman, et al. PCT WO 95/15749 published Jun. 15,1995.

The corresponding methyl ketones may be prepared by hydrogenolysis ofthe chloromethyl ketones as described by Kettner, et al. U.S. Pat. No.4,652,552.

When Q is an alpha-difluoromethyl ketone, the corresponding1-amino-3,3-difluoro-2-propanol can be prepared and coupled by CouplingProcedure II, and the resulting difluoroalcohol oxidized to thedifluoroketone by the procedure described by Trainor and Stein, U.S.Pat. No. 4,923,890.

When Q is a trifluoromethyl ketone (i.e. where H₂ N--R₄ is an aminoacid-derived trifluoromethyl ketone, e.g. H₂ N--CH(CH₂ --R₇)--COCF₃),the requisite ketone may be prepared as described by Imperiali andAbeles, Biochemistry 25, 3760 and supplementary pages (1986).

When Q is a monofluoromethyl ketonet (i.e. where H₂ N--R₄ contains afluoromethyl ketone group, e.g. H₂ N--CH(CH₂ --R₇)--COCH₂ F) derivedfrom an amino acid, the requisite ketone may be prepared by theprocedure of Falmer in European Patent Application EP 442,754 using theBoc-protected amino acid and magnesium benzyl fluoromalonate. Afterremoval of the Boc protecting groups, the amine can be coupled usingCoupling Procedure II. Alternatively, the monofluoromethyl ketone can beprepared by oxidation of the corresponding alcohol as described inExample 42.

When Q is a boroamino acid derivative (i.e. Q=--B(OH)₂ or its cyclicester), the cyclic ester is prepared essentially as described by Shenvi,U.S. Pat. No. 4,537,773, and after coupling by Coupling Procedure II,can optionally be converted to the free peptide boronic acid asdescribed by Shenvi and Kettner in U.S. Pat. Nos. 4,499,082 and5,187,157, and in J. Biol. Chem. 259, 15106 (1984).

When Q is an alpha-diketone or alp;ha-keto ester (i.e. Q=--C(═O)C(═O)R₇or --C(═O)CO₂ R₇) the requisite amino diketones or alpha-keto esters canbe prepared as described by Angelastro, et al, J. Med. Chem. 33, 13(1990) and references therein. The alpha-kimto esters may be hydrolyzedor amidated to produce the corresponding alpha-keto acids or amides. Thealpha-keto amides may also be prepared from commercially availableBoc-protected amino acids by a modification of the procedure ofHarbeson, et al. J. Med. Chem. 37, 2918-2929 (1994). In this latter veryuseful procedure, the Boc-protected amino acid is sequentially convertedto an N,O-dimethylhydroxylamide, an aldehyde, a cyanohydrin, analpha-hydroxycarboxylate, and an alpha-hydroxycarboxamide:

    R.sup.1 --COOH→R.sup.1 --CON(CH.sub.3)--OCH.sub.3 →R.sup.1 --CHO→R.sup.1 --CH(OH)--CN→R.sup.1 --CH(OH)--COOH→R.sup.1 --CH(OH)--CONH--R

where R¹ =Boc--NH--CH(CH₂ R₇)--.

The Boc group is removed under acidic conditions and afterneutralization, the free amino residue is coupled as in CouplingProcedure II to yield the alpha-hydroxycarboxamide which is thenoxidized to give the alpha-ketocarboxamide inhibitor:

    Boc--NH--CH(CH.sub.2 R.sub.7)--CH(OH)--CONH--R.sub.7 →

    H.sub.2 N--CH(CH.sub.2 R.sub.7)--CH(OH)--CONH--R.sub.7 --(Coupling Procedure II)→

    R.sub.1 --(CH.sub.2)--CHR.sub.2 --CONH--CH(R.sub.3)--CONH--CH(CH.sub.2 R.sub.7)--CH(OH)--CONHR.sub.7 →

    R.sub.1 --(CH.sub.2).sub.n --CH(R.sub.2)--CONH--CH(R.sub.3)--CONH--CH(CH.sub.2 R.sub.7)--COCONH--R.sub.7

EXAMPLE 7

Method A: Mixed anhydride method

Scheme ##STR7##

X=Fluorenylmethyloxycarbonyl (Fmoc)- or t-butyloxycarbonyl (Boc)- group;Y=Acetal or other protecting group for the amino aldehyde

AA₁ =Amino acid

AA₂ =Amino aldehyde

N-methylmorpholine (NMM) (1 eq.) was added to a stirred solution of theprotected amino acid in tetrahydrofuran (THF). The mixture was cooled to-15° C., treated with isobutyl chloroformate (IBCF) (1.1 eq.), andallowed to react for 10 min. Subsequently, the amino aldehyde componentin the form of the free base was added followed by NMM (1.1 eq., 2.2 eq.if acid salt). Stirring proceeded for 30 min at -15° C., and then atroom temperature for 3-4 h. The reaction mixture was dissolved in150-200 mL of ethyl acetate (EtOAc). The resulting solution wassuccessively washed with water, 5% sodium hydrogen carbonate (NaHCO₃),2% aqueous citric acid and, finally, water. The organic layer was driedover anhydrous sodium sulfate (Na₂ SO₄) or magnesium sulfate (MgSO₄),the solvent was removed under reduced pressure, and the product thusobtained was triturated with petroleum ether. The solid peptide thusobtained was filtered off, dried and characterized.

EXAMPLE 8

Method B: Fmoc-group deprotection procedure

Scheme

    Fmoc--HN--AA.sub.1 --AA.sub.2 --Y→H.sub.2 N--AA.sub.1 --AA.sub.2 --Y

The Fmoc-protected peptide (0.2 to 1 mmol) in a mixture of 30%dimethylformamide (DMF) in ethyl acetate (EtOAc) was treated with a50-60 fold excess of diethylamine (DEA) for 2 h at room temperature. Thesolvent was evaporated at reduced pressure at. 30° C., and petroleumether was added to the residue. When a precipitate formed it wasfiltered and dried. In other cases, the resulting gum was repeatedlytriturated with petroleum ether, and the gum was stored under vacuum.

EXAMPLE 9

Method C: Capping group or amino acid addition to the peptide

Scheme

    R--COOH+H.sub.2 N--(AA).sub.n --COOR'→R--CO--HN--(AA).sub.n --COOR'

The capping group (as a free carboxylic acid) or protected amino acid (1eq.), benzotriazol-1-yloxy-tris-(dimethyamino)-phosphoniumhexafluorophosphate (BOP) (1.1 eq.) and 1-hydroxybenzotriazole (HOBt) (1eq.) were dissolved in 5 mL of DMF followed by NMM (2.2 eq.). After 5min, the deprotected basic component of the peptide or the carboxylgroup protected amino acid was added, the pH was adjusted to 8 and themixture was stirred for 3-4 h. It was then diluted with 150-300 mL ofEtOAc and extracted consecutively with water, 2% NaHCO₃, water, 2%citric acid and water. The organic phase was dried and evaporated todryness to yield a capped or N-protected peptide.

EXAMPLE 10

Method D: Carbobenzyloxy (CBZ-) group removal procedure

A solution of the CBZ-protected peptide or amino acid derivative (1 g)in ethyl acetate (15 mL) was mixed with 0.2 g of Pd/C on carbon (10% Pdon carbon containing 50% by weight of water) and hydrogenated for 4 h at40 psi. The solution was filtered through Celitee (diatomaceous earth)and evaporated to dryness to yield the unprotected peptide or amino acidderivative.

EXAMPLE 11

Method E: Conversion of acetal to aldehyde

A solution of the peptide acetal (1. eq.) in 3 mL of THF was mixed with3 mL of aqueous HCl (2M) and stirred for 0.5-2 h. The solvent wasremoved by evaporation and the final residue was diluted with water andlyophilized to give the peptide aldehyde.

EXAMPLE 12

Method F: Preparation of Leucinal diethylacetal ##STR8## Step 1: NMM (10mL) was added to a solution of CBZ-Leu-OH (25 g, 93 mmol) in 250 mL ofTHF. The solution was cooled to -15° C., treated with 13 mL of isobutylchloroformate (IBCF), and allowed to react for 10 min. Subsequently, asuspension of N,O-dimethylhydroxylamine hydrochloride (9.36 g, 96 mmol)in 40 mL of DMF and 10 mL of NMM was added. After 4 h of stirring, themixture was diluted with 400 mL of EtOAc and the solution wasconsecutively washed with water, 5% NaHCO₃ solution, water, 2% citricacid, and water. The organic layer was dried over MgSO₄ and evaporatedto yield 19.0 g of a colorless oil.

Step 2: A solution of the oil (19 g) from step 1, in 200 mL of ether wascooled to -78° C., and 120 mL of a solution of lithium aluminium hydride(LiAlH₄) (1.0M) in ether was added dropwise over a period of 45 min. Thesolution was stirred for 30 min, and 200 mL of 1M potassium hydrogensulfate (KHSO₄) was added dropwise under a nitrogen atmosphere. Theorganic layer was separated, washed with KHSO₄ (1M) solution, dried(MgSO₄) and evaporated to a colorless liquid (CBZ-Leu-H).

Step 3: 104 mL of triethyl orthoformate was added over a period of 30min to a solution of CBZ-Leu-H (12 g) and p-toluenesulfonic acid (0.9 g)in 100 mL of anhydrous ethanol (EtOH). The mixture was stirred for 30min, and then was evaporated and diluted with 500 mL of ether. The etherlayer was washed consecutively with saturated solutions of NaHCO₃ andsodium chloride. The yellowish brown semisolid was recrystallized fromcold hexane to yield off-white needles of CBZ-Leucinal diethylacetal. ¹H NMR (300 MHz, CDCl₃) δ: 7.28(m, 5H), 5.03(s, 2H), 4.77(d, 1H), 4.27(d,1H), 3.8(m, 1H), 3.63(m, 2H), 3.43(m, 2H), 1.6(dd, 2H), 1.30(m, 2H),1.12(m, 6H), 0.83(d, 6H)

Step 4: The product (14.8 g) from step 3 was hydrogenated using methodD, with 2.5 g of Pd/C to yield 4.09 g of leucinal diethylacetal as anoil. ¹ H NMR (300 MHz, CDCl₃) δ: 4.16(d, 1H), 3.70(m, 3H), 3.75(m, 2H),2.87(m, 1H), 1.78(m, 1H), 1.33(m, 2H), 1.23(m, 6H), 0.935(dd, 6H)

EXAMPLE 13

Method G: Synthesis of P3 mimics (For nomenclature, see Schechter, I.and Burger, A. (1967) Biochem. Biophys. Res. Commun. 27: 157-162).##STR9## Step 1: Preparation of benzyl cyclopentaneacetate:

A mixture of cyclopentylacetic acid (10.02 g, 78.2 mmol), benzyl alcohol(8.45 g, 78.2 mmol) and p-toluenesulfonic acid monohydrate (1.48 g,7.82mmol) in benzene (60 mL) was refluxed using a Dean-Stark water separatorfor 2 h. After cooling, benzene was removed and the mixture was dilutedwith ether (50 mL) and washed successively with saturated NaHCO₃solution, saturated brine solution, dried and evaporated to give thecompound (15.40 g) as an oil; ¹ H NMR (300 MHz, CDCl₃) δ: 7.35(m, 5H),5.10(s, 2H), 2.40(d, j=8 Hz, 2H), 2.26(m, 1H), 1.81(m, 2H), 1.6(m, 4H),1.18(m, 2H).

Step 2: Preparation of benzyl 2-cyclopentyl-10-iododecanoate

To a cooled (-78° C.) solution of lithium diisopropylamide (20 mmol) ina mixture of THF (20 mL) and hexane (8 mL) (obtained in situ from thecorresponding diisopropylamine and n-BuLi) was added slowly the compoundobtained in step 1(3.96 g, 18 mmol) in anhyd. THF(10 mL). The mixturewas stirred for 30 minutes and 1,8-diiodooctane (7.19g, 20 mmol) inhexamethylphosphoramide (3.50 g, 20 mmol), was added. The mixture wasstirred at -78° C. for 30 minutes, slowly brought to 0° C. over a periodof 2 h and quenched by the cautious addition of 50 mL of 12% aqueoussodium chloride solution. The mixture was extracted with ether, washedwith brine, dried and the solvent was evaporated. The crude product waspurified to an oil (3.23 g) by a flash chromatography over silica usinghexane to 1% EtOAc in hexane as an eluant; ¹ H NMR (300 MHz, CDCl₃) δ:7.35(m, 5H), 5.15(s, 2H), 3.20(t, 8 Hz, 2H), 2.20(m, 1H), 2.0(m, 1H),1.2-1.8(m, 22H)

Step 3: Preparation of benzyl 10-cyano-2-cyclopentyldecanoate

A mixture of the iodoester from step 2, (3.99 g, 8.7 mmol) and sodiumcyanide (0.47 g, 9.6 mmol) in 15 mL of anhyd. DMSO was heated at 70-75°C. for 30 min. After cooling, the reaction mixture was poured over ice(.sup.˜ 40 g), extracted into ether and washed successively with waterand saturated brine. The organic layer was concentrated to yield an oil(2.89 g). ¹ H NMR (300 MHz, CDCl₃). δ: 7.35 (m, 5H), 5.15 (s, 2H),2.30(t, 8 Hz, 2H), 2.20(m, 1H), 2.00(m, 1H), 1.3-1.8 (m, 22H).

Step 4: Preparation of benzyl 10-N-phthalimido-2-cyclopentyldecanoate

A mixture of the compound from step 2(1.21 g, 2.6 mmol)and potassiumphthalimide (0.536 g, 3 mmol) in 8 mL of DMF was heated at 70-750 for 30min. After cooling, the reaction mixture was poured over ice (.sup.˜ 40g) and extracted into 60 mL of ether. The combined organic layer aswashed with water and saturated brine and concentrated to a colorlessoil (1.24 g). ¹ H NMR (300 MHz, CDCl₃) δ : 7.85(m, 2H), 7.70(m, 2H),7.35(m, 5H), 5.10(s, 2H), 3.65(t, 8 Hz, 2H), 2.20(m, 1H), 2.00(m, 1H),1.22-1.18(m, 22H).

Step 5: Preparation of 10-cyano-2-cyclopentyldecanoic acid

A mixture of the cyanoester from step 3 (2.89 g, 8 mmol) and 10%, Pd--C(0.6 g, DeGussa, H₂ O content 50%) in anhyd MeOH (35 mL) washydrogenated for 2 h (42-26 psi). The reaction mixture was filteredthrough a Celite® pad and concentrated to a colorless oil (2.02 g). ¹ HNMR (300 MHz, CDCl₃) δ 2.35(t, 8 Hz, 2H), 2.20 (m, 1H), 2.00 (m, 1H),1.3-1.9(m, 22H).

Step 6: Preparation of 2-cyclopentyl-10-N-phthalimidodecanoic acid

Following the procedure of step 5, product (0.41 g) of step 4 wasconverted to the title compound (0.31 g). ¹ H NMR (300 MHz, CDCl₃) δ7.85 (m, 2H), 7.70 (m, 2H), 3.70(t, 8 Hz, 2H), 2.15(m, 1H), 1.95(m, 1H),1.10-1.85(m, 22H).

Step 7: Preparation of10-trifluoromethanesulfonamido-2-cyclopentyl-decanoic acid benzyl ester##STR10## A mixture of the ester from step 4(0.874 g, 1.7 mmol) andhydrazine monohydrate (0.425 g, 0.41 mL, 8.5 mmol) in methanol (8 mL)was heated under reflux for 30 min, concentrated, and the residue wastriturated with ether to give a white precipitate which was filteredoff. The filtrate was concentrated to give an oil (0.540 g). The oil wasdissolved in CH₂ Cl₂ (8 mL) cooled to -10° C. and to it was addedtriethylamine (0.324 mL) followed by trifluoromethane sulfonylchloride(0.25 mL). The temperature was slowly brought to room temperature over aperiod of 1 h. The reaction mixture was then washed with water, 2% HCl,water and saturated brine. The organic layer was concentrated to an oiland was purified by flash chromatography over silica gel using 10% EtOAcin hexane as an eluant to yield10-trifluoromethanesulfonamido-2-cyclopentyl-decanoic acid benzyl esteras an oil (0.25 g). ¹ H NMR (300 MHz, CDCl₃) δ 7.35(m, 5H), 5.15(s,broad, 1H), 5.10(s, 2H), 3.25(m, 1H), 2.20(m, 1H), 2.00(m, 1H),1.10-1.7(m, 22H).

Step 8: Preparation of2-cyclopentyl-10-trifluoromethanesulfonamido)-decanoic acid

Following the same procedure as described in step 5, compound (0.25 g)obtained from step 7 was converted to the title compound (0.20 g) as anoil. ¹ H NMR (300 MHz, CDCl₃) δ 6.60-5.60 (b, 1H), 3.30 (d, 8 Hz, 2H),2.15(m, 1H), 1.90 (m, 1H), 1.1-1.8 (m, 22H). ##STR11## Step 9:Preparation of t-butyl 8-iodocaprylic acid

The title compound was synthesized following the procedure as describedfor the compound in step 2 from t-butyl acetate (1.16 g) and1,6-diiodohexane (4.06 g) as an oil (2.13 g). ¹ H NMR (300 MHz, CDCl₃) δ3.20(t, 8 Hz, 2H), 2.20(t, 8 Hz, 2H), 1.75-1.85(m, 2H), 1.55-1.65(m,2H), 1.45(s, 9H), 1.25-1.43(m, 6H).

Step 10: Preparation of 2-cyclopentyldecan-1,10-dioicacid-1-benzyl-10-t-butyl ester

Using the procedure as described in step 2, the ester (6.92 g) from step1 and the iodoester (11.37 g) from step 9 were reacted to give the titlecompound (7.44 g) oil. ¹ H NMR (300 MHz, CDCl₃) δ 7.35 (m, 5H), 5.10 (s,2H), 2.20(m, 3H), 2.00(m, 1H), 1.45 (s, 9H), 1.0-1.9 (m, 20H)

Step 11: Preparation of 2-cyclopentyldecan-1,10-dioicacid-1-benzyl-10-methyl ester

A solution of the diester from step 10 (2.86 g, 7 mmol) in CH₂ Cl₂ (30mL) and 10 mL of 90% TFA was stirred for 30 min. The product obtainedafter evaporation was dissolved in a mixture of CH₂ Cl₂ (10 mL) and MeOH(6 mL). The solution was stirred at -20° C. and to it was slowly addedthionyl chloride (6 mL) over a period of 2 h. The solvent was evaporatedand the residue was dissolved in diethyl ether (20 mL). The organiclayer was washed successively with saturated NaHCO₃ solution, water andbrine, dried, evaporated and flash chromatographed on silica gel(eluant: 2% EtOAc-hexane) to furnish the product as an oil (2.05 g). ¹ HNMR (300 MHz, CDCl₃) δ 7.35 (m, 5H), 5.10(s, 2H), 3.65 (s, 3H), 2.30(t,8 Hz, 2H), 2.20 (m, 1H), 02.00 (m, 1H), 1.10-1.90(m, 10H)

Step 12: Preparation of 2-cyclopentyldecan-1,10-dioic acid-10-methylester

Using the procedure as described in step 5, the compound (4.96 g) fromstep 11 was converted to the title compound (3.66 g) as an oil. ¹ H NMR(300 MHz, CDCl3) δ 3.65(s, 3H), 2.30(t, 8 Hz, 2H), 2.15(m, 1H), 2.00(m,1H), 1.10-1.19(m, 20H).

Step 13: Preparation of 6-cyanohexane-1-sulfinic acid sodium salt##STR12## In a three necked flask, fitted with a water condenser, amechanical stirrer and a dropping funnel a solution of1-bromo-6-cyanohexane (8.04 g, 42.3 mmol) was placed in 75 mL of 95%EtOH. The mixture was heated to reflux and to it was slowly added asolution of sodium sulfite (8.00 g, 63.5 mmol) in 50 mL of H₂ O over aperiod of 30 min. The mixture was heated for 2 h and then concentratedunder reduced pressure. The residue was dissolved in 95% EtOH (200 ml),heated to boiling and filtered. The filtrate was concentrated and cooledin an ice bath. The precipitate was collected by filtration and dried togive 3.00 g of a white solid. The mother liquor on furtherconcentration, produced another batch of 2.2 g of the product. ¹ H NMR(300 MHz, DMSO-d₆) δ 2.50 (t, j=8 Hz, 2H), 2.40(t, 8 Hz, 2H), 1.55(m,4H), 1.3 (m, 4H).

Step 14: Preparation of 6-cyanohexane-1-sulfonyl chloride

A mixture of the product from step 13 (0.340 g, 1.6 mmol), thionylchloride (0.95 g, 8 mmol) and a drop of DMF was heated at 75-80° C. for30 min. After cooling, the solvent was removed and the residue wastreated with ice-water (5 mL). The organic layer was extracted into 30mL of ether and the combined organic layer was washed with 3% NaHCO₃solution and water, dried (MgSO₄) and filtered. The filtrate was treatedwith active carbon (Darco), filtered and concentrated to give acolorless oil (0.240 g) which was used without further purification.

Examples 14-38 describe the syntheses of the MCP inhibitors listed inTable 5. The corresponding purification conditions are listed in Table6.

                                      TABLE 5                                     __________________________________________________________________________    Multicatalytic Protease Inhibitors                                            __________________________________________________________________________    1 #STR13##                                                                    Example No.                                                                          R       a W      X   Y        Z       IC.sub.50 nM                     __________________________________________________________________________    14     NC--    8                                                                               2 #STR14##                                                                           NO.sub.2                                                                          (CH.sub.3).sub.2 CH--                                                                  O       7/6                              15     NC--    8 "      PMC (CH.sub.3).sub.2 CH--                                                                  O       20                               16     NC--    8 "      NO.sub.2                                                                          (CH.sub.3).sub.2 CH--                                                                  N--NHCONH.sub.2                                                                       100                              17     NC--    8 "      NO.sub.2                                                                          (CH.sub.3).sub.2 CH--                                                                  N--OH   . . .                            18     NC--    8 "      NO.sub.2                                                                          (CH.sub.3).sub.2 CH--                                                                  N--OCH.sub.3                                                                          700                              19     NC--    8 "      NO.sub.2                                                                          (CH.sub.3).sub.2 CH--                                                                  N--O--CH.sub.2 C.sub.6 H.sub.5                                                        . . .                            20     MeOOC-- 7 "      PMC (CH.sub.3).sub.2 CH--                                                                  O       10                               21     MeOOC-- 7 "      MTR (CH.sub.3).sub.2 CH--                                                                  O       30                               22     MeOOC-- 7 "      PMC CH.sub.3 --CH.sub.2 CH.sub.2 --                                                        O       110                              23     MeOOC-- 7 "      NO.sub.2                                                                          (CH.sub.3).sub.2 CH--                                                                  O       5                                24     C.sub.6 H.sub.4 (CO).sub.2 N--                                                        8 "      NO.sub.2                                                                          (CH.sub.3).sub.2 CH--                                                                  O       2                                25     CF.sub.3 --SO.sub.2 NH--                                                              8 "      NO.sub.2                                                                          (CH.sub.3).sub.2 CH--                                                                  O       8/5                              26     MeOOC-- 6 H      NO.sub.2                                                                          (CH.sub.3).sub.2 CH--                                                                  O       40                               27     MeOOC-- 6 H      NO.sub.2                                                                          C.sub.6 H.sub.5                                                                        O       130                              28     MeOOC-- 6 H      PMC (CH.sub.3).sub.2 CH--                                                                  O       20                               29     MeOOC-- 6 H      PMC (CH.sub.3).sub.2 CH--                                                                  O       >300                             30     MeOOC-- 6 H      PMC CH.sub.3 --CH.sub.2 CH.sub.2 --                                                        O       >100                             31     MeOOC-- 6 H      TOS (CH.sub.3).sub.2 CH--                                                                  O       75                               32     MeOOC-- 6 H      MTR (CH.sub.3).sub.2 CH--                                                                  N--NHCONH.sub.2                                                                       220                              33     MeOOC-- 6 H      MTR (CH.sub.3).sub.2 CH--                                                                  N--NHCSNH.sub.2                                                                       700                              34     MeOOC-- 6 H      MTR (CH.sub.3).sub.2 CH--                                                                  O       80                               35     MeOOC-- 6 H      MTS (CH.sub.3).sub.2 CH--                                                                  O       40                               __________________________________________________________________________    3 #STR15##                                                                    4 #STR16##                                                                    5 #STR17##                                                                    Example No.                                                                         R        m n W X                 Y      IC.sub.50                       __________________________________________________________________________                                                  nm                              36    NC--     5 1 H PMC--NH--C(═NH)--NH--(CH.sub.2).sub.3 --                                                    --SO.sub.2 --                                                                        >>100                           37    β-Naphthyl                                                                        0 0 --                                                                              O.sub.2 N--NH--C(═NH)--NH--(CH.sub.2).sub.3                                                 6 #STR18##                                                                           250                             38                                                                                  7 #STR19##                                                                             5 1 H O.sub.2 N--NH--C(═NH)--NH--(CH.sub.2).sub.3                                                 6 #STR20##                                                                           . . .                           __________________________________________________________________________

                                      TABLE 6                                     __________________________________________________________________________                                 HPLC gradient                                                                            Analyt.                                                            (Ret. time of the                                                                        rt. time                              Example No.                                                                          COMPOUNDS             peak collected)                                                                          in min.                                                                            Mol. Wt                                                                             (M + H).sup.+              __________________________________________________________________________    14     10-cyano-2-cyclopentyldecanoyl-N.sup.g -nitro-L-                                                    45-75% B (10.63)                                                                         22.5 563.75                                                                              564                               arginyl-L-leucinal                                                     15     10-Cyano-2-cyclopentyl-decanoyl-N.sup.g -                                                           43-100% B (22.0)                                                                         31.7 785.2 786                               (2,2,5,7,8-pentamethylchroman-6-sulfonyl)-                                    L-arginyl-L-leucinal                                                   16     10-cyano-2-cyclopentyl-decanoyl-N.sup.g -nitro-L-                                                   30-35% B 60 min                                                                          22.2 621   621                               arginyl-L-leucinal semicarbazone                                                                    (A = 30.0, B = 32.0)                                                                     22.7                                  17     10-cyano-2-cyclopentyl-decanoyl-N.sup.g -nitro-L-                                                   45-65% B (12.3)                                                                          24.8 578.7 579                               arginyl-L-leucinal oxime                                               18     10-cyano-2-cyclopentyl-decanoyl-N.sup.g -nitro-L-                                                   55-75% B   27.7 592.7 593                               arginyl-L-leucinal-O-methyloxime                                                                    (A = 8.9, B = 10.12)                             19     10-Cyano-2-cyclopentyl-decanoyl-N.sup.g -nitro-L-                                                   65-75% B   31.3 668.7 669                               arginyl-L-leucinal-O-benzyloxime                                                                    (A = 8.3, B = 9.5)                               20     9-Methoxycarbonyl-2-cyclopentyl-nonanoyl-N.sup.g -                                                  60-100% B (14.1)                                                                         32.0 804.11                                                                              805                               (2,2,5,7,8-pentamethylchroman-6-sulfonyl)-L-                                  arginyl-L-leucinal                                                     21     9-Methoxycarbonyl-2-cyclopentyl-nonanoyl-N.sup.g -                                                  40-60% B (29.9)                                                                          28.9 749   750                               (4-methoxy-2,3,6-trimethyl-benzene-1-sulfonyl)-                               L-arginyl-L-leucinal                                                   22     9-Methoxycarbonyl-2-cyclopentyl-nonanoyl-N.sup.g -                                                  60-70% B (18.8)                                                                          32.2 804   804                               (2,2,5,7,8-pentamethylchroman-6-sulfonyl)-L-                                  arginyl-L-norleucinal                                                  23     9-Methoxycarbonyl-2-cyclopentyl-nonanoyl-N.sup.g -                                                  40-70% B (13.98)                                                                         23.6 582.34                                                                              583                               nitro-L-arginyl-L-leucinal                                             24     2-cyclopentyl-10-N-phthalimido-decanoyl-N.sup.g -                                                   50-80% B (12.66)                                                                         27.89                                                                              683.86                                                                              684                               nitro-L-arginyl-L-leucinal                                             25     10-(trifluoromethanesulfonyl)amino-2-                                                               50-70% B   27.20                                                                              686.02                                                                              686                               cyclopentyl-decanoyl-N.sup.g -nitro-L-arginyl-L-                                                    (A = 12.1, B = 12.9)                                                                     27.46                                        leucinal                                                               26     Monomethylazelayl-N.sup.g -nitro-L-arginyl-L-                                                       30-100% B (8.14)                                                                         17.06                                                                              501   501                               leucinal                                                               27     Monomethylazelayl-N.sup.g -nitro-L-arginyl-L-                                                       10-100% B (16.9)                                                                         17.3 534   535                               phenylalaninal                                                         28     Monomethylazelayl-N.sup.g -(2,2,5,7,8-                                                              10-100% B (11.7)                                                                         26.5 722   723                               pentamethylchroman-6-sulfonyl)-L-arginyl-L-                                   leucinal                                                               29     Monomethylazelayl-N.sup.g -(2,2,5,7,8-                                                              40-70% B (16.2)                                                                          26.3 722   723                               pentamethylchroman-6-sulfonyl)-D-arginyl-L-                                   leucinal                                                               30     Monomethylazelayl-N.sup.g -(2,2,5,7,8-                                                              50-60% B (14.0)                                                                          26.2 722   723                               pentamethylchroman-6-sulfonyl)-L-arginyl-L-                                   norleucinal                                                            31     Monomethylazelayl-N.sup.g -(p-toluenesulfonyl)-L-                                                   40-50% B (19.6)                                                                          21.3 610   610                               arginyl-L-leucinal                                                     32     Monomethylazelayl-N.sup.g -(4-methoxy,2,3,6-                                                        --         13.2 724   725                               trimethyl-1-sulfonyl)-L-arginyl-L-leucinal                                    semicarbazone                                                          33     Monomethylazelayl-N.sup.g -(4-methoxy,2,3,6-                                                        --         23.4 741   741                               trimethyl-1-sulfonyl)-L-arginyl-L-leucinal                                    thiosemicarbazone                                                      34     Monomethylazelayl-N.sup.g -(4-methoxy,2,3,6-                                                        40-44% B (60 min)                                                                        23.3 668   669                               trimethylbenzene-1-sulfonyl)-L-arginyl-L-                                                           (16.9)                                                  leucinal                                                               35     Methoxyazelaoyl-N.sup.g -(2,4,6-trimethylbenzene-1-                                                 40-60% B (14.1)                                                                          32.0 638   638                               sulfonyl)-L-arginyl-L-leucinal                                         36     6-Cyano-hexane-1-sulfonyl-N.sup.g -(2,2,5,7,8-                                                      45-75% B (13.74)                                                                         24.94                                                                              710.9 711                               pentamethylchroman-6-sulfonyl)-L-arginyl-L-                                   leucinal                                                               37     2-Naphthoyl-N.sup.g -nitro-L-arginyl-L-leucinal                                                     35-55% B (9.16)                                                                          18.10                                                                              470.51                                                                              471                        38     CBZ-7-aminoheptanoyl-N.sup.g -nitro-L-arginyl-L-                                                    30-60% B   19.1 577   578                               leucinal              (A = 13.3, B = 13.9)                             __________________________________________________________________________     HPLC conditions:                                                              Solvent A: Water containing 0.1% TFA                                          Solvent B: Acetonitrile containing 0.1% TFA                                   Detection: 215 nm.                                                            HPLC gradients are in 40 min. unless noted otherwise.                         Column Type:                                                                  Zorbax R.sub.x --C.sub.8 -                                                    Dimension 4.6 mm × 250 mm                                               Pore Size 80 Angstroms                                                        Particle Size 5 micron                                                   

EXAMPLE 14 10-cyano-2-cyclopentyldecanoyl-N^(g)-nitro-L-arginyl-L-leucinal ##STR21## Step 1:Fluorenylmethyloxycarbonyl-N^(g) -nitro-L-arginyl-L-leucinaldiethylacetal.

Fmoc-Arg(NO₂)-OH (4.41g, 10 mmol) was coupled with Leu-acetal (1.89g, 10mmol) according to method A (Example 7), using 1.29 mL of IBCF, 2.2 mLof NMM and 10 mL of THF (in the place of DMF as in method A of Example7). The crude peptide was obtained as amorphous solid. ¹ H NMR (300 MHz,CDCl₃) δ 7.75 (d, 2H), 7.55 (d, 2H), 7.4 (m, 2H), 7.29(m, 2H), 6.21(d,1H), 5.91(d, 1H), 4.30(m, 5H), 3.66(m, 3H), 3.63(d, 2H), 3.32(m,2H),1.72(m, 4H), 1.29(m, 2H), 1.17(b, 6H), 0.89 (m, 6H).

Step 2: N^(g) -nitro-L-arginyl-L-leucinal diethylacetal

The Fmoc group was removed from 3.5 g of the product from step 1 usingdeprotection method B (Example 8). The free base (2.5 g) was obtained assemisolid. ¹ H NMR (300 MHz, DMSO-d₆) δ 8.6 (b, 1H), 7.61(d, 1H), 4.27(d, 1H), 3.90(m, 1H), 3.59(m, 1H), 3.45(m, 4H), 3.14(m,2H), 1.54 (m,4H), 1.33(m, 3H), 1.10(tt, 6H), 0.83(dd, 6H).

Step 3: 10-cyano-2-cyclopentyl-decanoyl-N^(g)-nitro-L-arginyl-L-leucinal diethylacetal.

According to method C (Example 9), 10-cyano-2-cyclopentyl-decanoic acid(2 g, 7.5 mmol) from method G, in 16 mL of DMF was treated with productof Step 2 (2.15 g, 5 mmol) using BOP (3.34 g, 7.5 mmol) and HOBt (1.01g, 7.5 mmol) to yield the crude peptide (3.85 g) as a pale yellow solid.¹ H NMR (300 MHz, DMSO-d6) δ 8.31 (bd, 1H), 8.0 (bd, 1H), 7.83 (d, 1H),4.34(m, 1H), 4.24(d, 1H), 3.91(m, 1H), 3.56(m, 4H), 3.43(m, 2H), 3.16(m,2H), 2.47(t, 3H), 2.03(m, 1H), 1.76(m, 2H), 1.15-2.0(m, 25H), 1.07(m,6H), 0.81(d, 6H)

Step 4: A solution of the product of step 3 (0.2 g) in 10 mL ofacetonitrile (ACN) containing 30% TFA was stirred for 1 h, and thesolvent was evaporated and Compound 14 was precipitated using diethylether. The HPLC purification conditions are given in Table 6. ¹ HNMR(300 MHz, DMSO-d₆) δ9.38(s, 1H), 8.56(b, 1H), 8.3(d, 1H), 7.99(dd,1H), 4.38(m, 1H),4.17(m, 1H), 3.37(m, 4H), 3.17(m, 2H), 2.26(t, 3H),1.0-2.0(m, 27H), 0.86(dd, 6H).

EXAMPLE 15 10-Cyano-2-cyclopentyl-decanoyl-N^(g)-(2,2,5,7,8-pentamethylchroman-6-sulfonyl)-L-arginyl-L-leucinal##STR22## Step 1: 10-Cyano-2-cyclopentyl-decanoyl-N^(g)-(2,2,5,7,8-pentamethylchroman-6-sulfonyl)-L-arginyl-L-leucinaldiethylacetal.

Following method C (Example 9), 10-cyano-2-cyclopentyl-decanoic acid (2mmol, from step 5 in method G of Example 13) was coupled with N^(g)-(2,2,5,7,8-pentamethylchroman-6-sulfonyl)-L-arginyl-L-leucinaldiethylacetal (1.8 mmol, from Step 2 in Compound 14) to obtain thecompound as solid.

Step 2: 10-Cyano-2-cyclopentyl-decanoyl-N^(g)-(2,2,5,7,8-pentamethylchroman-6-sulfonyl)-L-arginyl-L-leucinal.

Following Method E (Example 11), the product from step 1(0.3 g) wasconverted to Compound 15 (0.2 g) and purified by HPLC. ¹ H NMR(300 MHz,DMSO-d6) δ 9.38(s, 1H), 8.23(d, 1H), 7.8(s, 1H), 7.72(d, 1H), 7.49(t,1H), 7.48(s, 1H), 4.34(m, 1H), 4.14(m, 1H),3.05 (m, 2H), 2.60(t, 2H),2.50(s, 3H), 2.46(s, 3H), 2.25(m, 4H), 2.00(s, 3H), 1.74(t, 2H), 1.6-1.0(m, 25H), 1.13(s, 6H), 0.82 (dd, 6H)

EXAMPLE 16 10-cyano-2-cyclopentyl-decanoyl-Ng-nitro-L-arginyl-L-leucinal semicarbazone ##STR23##

Semicarbazide hydrochloride (0.20 mmol), sodium acetate (0.3 mmol) andwater (0.5 mL) were added to a solution of Compound 14 (0.050 g, 0.089mmol) in 4.5 mL of ethanol and stirred overnight at room temperature.Solvent was removed and the resulting Compound 16 was purified by HPLCas indicated in Table 6. ¹ H NMR (300 MHz, DMSO-d₆) δ 9.91(s, 1H),8.51(s, 2H) 8.04(d, 1H), 7.95(d, 1H)7.40(s, 1H), 7.09(d, 1H),6.29(s,2H),4.44(m, 1H), 4.33(m, 1H), 3.16(s, 2H), 2.08-1.00 (m, 33H), 0.87(dd,6H).

EXAMPLE 17 10-cyano-2-cyclopentyl-decanoyl-N^(g)-nitro-L-arginyl-L-leucinal oxime ##STR24##

Hydroxylamine hydrochloride (0.037 g, 0.534 mmol) was added to asolution of the product of step 4 in Example 14 (0.05 g, 0.089 mmol) inpyridine (0.175 g, 2.2 mmol) at room temperature and then at 80° C. for30 minutes. The product was isolated by HPLC as indicated in Table 6.

EXAMPLE 18 10-cyano-2-cyclopentyl-decanoyl-N^(g)-nitro-L-arginyl-L-leucinal-O-methyloxime ##STR25##

Following the procedure of Example 17, Compound 18 was prepared usingO-methylhydroxylamine hydrochloride (0.031 g, 0.38 mmol) and isolated asa mixture of two peaks by HPLC as indicated in Table 6.

EXAMPLE 19 10-Cyano-2-cyclopentyl-decanoyl-N^(g)-nitro-L-arginyl-L-leucinal-O-benzyloxime ##STR26##

Following the procedure as in the preparation of Example 17, Compound 19was made using Compound 14 (0.05 g, 0.089 mmol), O-benzyl hydroxylaminehydrochloride (0.043 g, 0.267 mmol) and pyridine (0.092 mL, 1.14 mmol).The compound was separated as two peaks in HPLC purifications.

EXAMPLE 20 9-Methoxycarbonyl-2-cyclopentyl-nonanoyl-N^(g)-(2,2,5,7,8-pentamethylchroman-6-sulfonyl)-L-arginyl-L-leucinal##STR27## Step 1: Fmoc-N^(g)-(2,2,5,7,8-pentamethylchroman-6-sulfonyl)-L-argininyl-L-leucinaldiethylacetal.

Following method C (Example 9), Fmoc-N^(g)-(2,2,5,7,8-pentamethylchroman-6-sulfonyl)-L-arginine (3.31 g, 5 mmol)was coupled with L-leucinal diethylacetal (0.85 g, from method F,Example 12) using BOP (2.21 g, 5 mmol), HOBt (0.67 g, 5 mmol) and NMM(0.7 mL) in 12 mL of DMF to yield the dipeptide acetal (3.24 g). ¹ H NMR(300 MHz,, CDCl₃) δ 7.89(s, 1H), 7.78 (d, 2H), 7.6 (d, 2H),7.4(t, 2H),7.31(t, 3H), 6.4(d, 2H),5.92(d, 1H), 4.58(m, 1H), 4.43(m, 1H), 4.35(m,3H), 3.69(m, 2H), 3.53(m, 2H), 3.30(m, 2H), 2.6(t, 2H), 2.58(s, 6H),2.12(s, 3H), 1.80(tt, 2H), 1.64(m, 3H), 1.32(m, 10H), 1.18(q, 6H),0.89(t, 6H).

Step 2: N^(g)-(2,2,5,7,8-pentamethylchroman-6-sulfonyl)-L-arginyl-L-leucinal.

Following method B (Example 8), the Fmoc group was removed from theproduct (1.6 g) from step 1, to yield a semi-solid (1.3 g). ¹ H NMR (300MHz, DMSO-d₆) δ 7.6(d, 1H), 6.76(b, 1H), 6.46(b, 1H), 4.27(d, 1H),3.9(m, 1H,), 3.58(m, 1H), 3.44(m, 4H), 3.11(t, 1H), 3.01(m, 2H), 2.58(t,2H), 2.47(s, 6H), 2.03(s, 3H), 1.77(t, 2H), 1.5(m, 5H), 1.26(s,6H),1.09(m, 6H), 0.83(dd, 6H)

Step 3: 9-Methoxycarbonyl-2-cyclopentyl-nonanoyl-N^(g)-(2,2,5,7,8-pentamethylchroman-6-sulfonyl)-L-arginyl-L-leucinaldiethylacetal.

Following method C (Example 9),9-methyloxycarbonyl-2-cyclopentyl-nonanoic acid (0.56 g) was coupledwith N^(g)-(2,2,5,7,8-pentamethylchroman-6-sulfonyl)-L-arginyl-L-leucinaldiethylacetal (0.7 g, 1.1 mmol) using BOP (0.66 g, 1.5 mmol), HOBt(0.202 g, 1.5 mmol) and NMM (2.2 mL, 2 mmol) in 5 mL of DMF to get theproduct (1.8 g) as solid. ¹ H NMR(300 MHz, MSO-d6) δ: 7.5-8.0(m, 2H),6.66(m, 1H), 6.4(m, 1H), 4.32(m, 1H), 4.24(d, 1H), 3.93(m, 1H), 3.57(m,6H), 3.44(m, 4H), 3.04(m, 2H), 2.59(t, 2H), 2.48(s, 6H), 2.28(m, 3H),2.03(s, 3H), 1.77(t, 2H), 1.48(m, 22H), 1.26(s, 6H), 1.1(tt, 9H),0.81(dd, 6H).

Step 4:9-Methoxycarbonyl-2-cyclopentyl-nonanoyl-Ng-(2,2,5,7,8-pentamethylchroman-6-sulfonyl)-L-arginyl-L-leucinal.

According to the method E (Example 11), the product (0.2 g) from step 1was converted to Compound 20 and purified by HPLC directly. ¹ H NMR(300MHz, DMSO-d₆) δ 9.37(s, 1H), 8.2-7.4(m, 2H), 6.69(drm, 1H), 6.4(brm,1H), 4.3(m, 2H), 3.58(s, 3H), 3.03 (m, 2H), 2.60(t, 2H), 2.48(s, 6H),2.26(m, 4H), 2.03(s, 3H), 1.77(t, 2H), 1.47(brm, 28H), 1.24(s, 3H), 0.80(dd, 6H).

EXAMPLE 21 9-Methoxycarbonyl-2-cyclopentyl-nonanoyl-N^(g)-(4-methoxy-2,3,6-trimethyl-benzene-1-sulfonyl)-L-arginyl-L-leucinal##STR28## Step 1: 9-Methoxycarbonyl-2-cyclopentyl-nonanoyl-N^(g)-(4-methoxy-2,3,6-trimethylbenzene-1-sulfonyl)-L-arginyl-L-leucinaldiethylacetal.

Following method C (Example 9), 9-Methoxycarbonyl-2-cyclopentyl-nonanoicacid (1.2 mmol, from step 12 in method G) was coupled with N^(g)-(4-methoxy-2,3,6-trimethylbenzene-1-sulfonyl)-L-arginyl-L-leucinaldiethylacetal (1.0 mmol) to yield the peptide as solid.

Step 2: Following method E (Example 11), the acetal from step 1 wasconverted to Compound 21 and purified by HPLC. ¹ H (300 MHz, CDCl₃/DMSO-d₆) δ 9.42(s, 1H), 8.25(d, 1H), 7.92(d, 1H), 7.84(s, 1H), 6.4(m,2H), 4.40(m, 1H), 4.20(m, 1H), 3.80(s, 3H), 3.57(s, 3H), 3.26(m, 2H),2.64(s, 3H), 2.55(s, 3H), 2.36(m, 2H), 2.07(s, 3H), 1.8-1.0(m, 30H),0.87(dd, 6H)

EXAMPLE 22 9-Methoxycarbonyl-2-cyclopentyl-nonanoyl-N^(g)-(2,2,5,7,8-pentamethylchroman-6-sulfonyl)-L-arginyl-L-norleucinal##STR29## Step 1: 9-Methoxycarbonyl-2-cyclopentyl-nonainoyl-N^(g)-(2,2,5,7,8-pentamethylchroman-6-sulfonyl)-L-arginyl-L-norleucinaldiethylacetal.

Using Method C (Example 9), 9-methoxycarbonyl-2-cyclopentyl-nonanoicacid (1 mmol) was coupled with N^(g)-(2,2,5,7,8-pentamethylchroman-6-sulfonyl)-L-arginyl-L-norleucinaldiethylacetal (0.69 mmol, obtained from step 1 in Example 30 followingmethod B) using BOP (1 mmol), HOBt (1 mmol) and NMM (2.5 mmol) in 5 mLof DMF. The reaction mixture was stirred for 4.5 hours and the peptidewas isolated (0.37 g, 0.42 mmol).

Step 2: 9-Methoxycarbonyl-2-cyclopentyl-nonanoyl-N^(g)-(2,2,5,7,8,-pentamethylchroman-6-sulfonyl)-L-arginyl-L-norleucinal.

According to Method E (Example 11), the product (0.37 g, 0.42 mmol) fromstep 1 was converted to Compound 22 (0.33 g) and purified by HPLC. ¹ HNMR(300 MHz, DMSO-d6) δ: 9.29(s, 1H), 7.79(d, 1H), 7.89(d, 1H), 7.31(t,1H), 7.26(s, 2H), 4.17(m, 1H), 3.94 (m, 1H), 3.49(s, 3H), 2.94 (m, 2H),2.74(t, 2H), 2.51(t, 2H), 2.37(s, 6H), 2.20(m, 4H), 1.94(s, 3H), 1.14(s,6H), 1.0-1.8 (m, 3H), 0.74 (t, 3H).

EXAMPLE 23 9-Methoxycarbonyl-2-cyclopentyl-nonanoyl-N^(g)-nitro-L-arginyl-L-leucinal ##STR30## Step 1:9-Methoxycarbonyl-2-cyclopentyl-nonanoyl-N^(g)-nitro-L-arginyl-L-leucinal diethylacetal.

Following Method C (Example 9), 9-methoxycarbonyl-2-cyclopentyl-nonanoicacid (0.66 g, 0.33 mmol) was coupled with the product from step 2 inExample 14 (0.109 g, 0.28 mmol) using BOP (0.146 g, 0.33 mmol), HOBt(0.0449 g, 0.33 mmol) and NMM (0.105, 0.95 mmol) in 7 mL of DMF to yieldthe title compound of Step 1 (0.124 g). ¹ H NMR(300 MHz, CDCl3 δ:8.31(m, 2H), 6.69(br d, 1H), 6.15(br d, 1H), 4.5(m, 1H), 4.33(m, 1H),4.18(m, 1H), 3.67(s, 3H), 3.53(m, 4H), 3.32(m, 2H), 2.68(m, 2H), 2.29(t,3H), 2.0-1.0(b m, 34H), 0.90(dd, 6H).

Step 2: Following method E (Example 11), the peptide (from step 1, 0.124g) was converted to Compound 23 (0.106 g). ¹ H NMR (300 MHz, DMSO-d₆).δ: 9.39(s, 1H), 8.54(m, 1H), 8.31(d, 1H), 7.99(br d,2H), 4.36(m, 1H),4.15(m, 1H), 3.33(s, 3H), 3.16(m, 2H), 2.27(t, 2H), 2.03(m, 2H),1.0-1.7(m, 28H), 0.83(dd, 6H).

EXAMPLE 24 2-cyclopentyl-10-N-phthalimido-decanoyl-N^(g)-nitro-L-arginyl-L-leucinal ##STR31## Step 1:2-cyclopentyl-10-N-phthalimido-decanoyl-N^(g)-nitro-L-arginyl-L-leucinal diethylacetal.

Following Method C (Example 9), the product (0.25 g, 0.65 mmol) fromstep 6 in Method G (Example 13) was coupled to the product from step 2in Example 14 (0.195 g, 0.5 mmol) using BOP (0.288 g, 0.65 mmol), HOBt(0.088 g, 0.65 mmol) and NMM (0.130 mL, 0.130 mmol) in 5 mL of DMF toobtain the title compound (0.557 g) as a solid. ¹ H NMR (300 MHz,DMSO-d6) δ: 8.57(br m, 1H), 7.91 (d, 1H), 7.85 (br d, 4H), 7.55 (d, 1H),4.32 (m, 1H), 4.23 (dd, 1H), 3.90(m, 1H), 3.55 (t, 3H), 3.43 (m, 4H),3.14(m, 2H), 2.0 (br m, 1H), 1.74 (br m, 2H), 2.0-1.15 (2 br m, 28H),1.09(tt, 6H), 0.79 (dd, 6H).

Step 2: 2-cyclopentyl-10-N-phthalimido-decanoyl-N^(g)-nitro-arginyl-L-leucinal.

Using method E (Example 11), the product of step 1(0.45 g) was convertedto Compound 24 (0.32 g). ¹ H NMR (300 MHz, DMSO-d₆) δ 9.38 (s, 1H), 8.31(d, 1H), 8.00 (d, 1H), 7.85 (m, 4H), 4.38 (m, 1H), 4.15 (1, 1H),3.57(tt, 3H), 3.15 (m, 2H), 2.00 (m, 1H), 1.9-1.0(br m, 30H), 0.86 (dd,6H).

EXAMPLE 2510-(trifluoromethanesulfonyl)amino-2-cyclopentyl-decanoyl-N^(g)-nitro-L-arginyl-L-leucinal ##STR32## Step 1:(Trifluoromethanesulfonyl)10-amino-2-cyclopentyl-decanoyl-N^(g)-nitro-L-arginyl-L-leucinal diethylacetal.

Using Method C (Example 9),10-(trifluoromethanesulfonyl)-amino-2-cyclopentyl-decanoic acid (0.199g, 0.51 mmol) was coupled to the product from step 2 in Example 14(0.175 g, 0.45 mmol) using BOP (0.22 g, 0.51 mmol), HOBt (0.069 g, 0.51mmol) and NMM (0.052 mL, 0.47 mmol) in 5mL of DMF. The acetal wasobtained as a solid (0.42 g). ¹ H NMR (300 MHz, CDCl₃) δ: 6.06(br m,1H), 5.81(m, 1H), 4.53(m, 1H), 4.32(d, 1H), 4.13(m, 1H), 3.73(q, 4H),3.53(m, 2H), 3.30(q, 3H), 2.0-1.0(2 br m, 36H), 0.9(dd, 6H).

Step 2: 10-(trifluoromethanesulfonyl)amino-2-cyclopentyl-decanoyl-N^(g)-nitro-L-arginyl-L-leucinal.

Following Method E (Example 11), the product from step 1 (0.35 g) wasconverted to Compound 25 (0.17 g). ¹ H NMR (300 MHz, DMSO-d₆) δ: 9.27(s,1H), 8.46(br m, 1H), 4.30(m, 1H), 3.87(m, 1H), 3.09(m, 5H), 2.8-1.0(2 brm, 30H), 0.78(dd, 6H).

EXAMPLE 26 Monomethylazelayl-N^(g) -nitro-L-arginyl-L-leucinal ##STR33##Step 1: Monomethylazelayl-N^(g) -nitro-L-arginyl-L-leucinaldiethylacetal:

Following Method C (Example 9), the title compound was made usingazelaic acid monomethyl ester (0.708 g, 3.5 mmol), BOP (1.55 g, 3.5mmol), HOBt (0.47 g, 3.5 mmol), NMM (0.38 mL, 3.5 mmol), N^(g)-nitro-L-arginyl-L-leucinal diethylacetal (obtained from step 2 inExample 14 following method B, Example B) (1.306 g, 3.5 mmol) in 12 mLof DMF. The peptide was obtained as an amorphous solid (2.17 g). ¹ H NMR(300 MHz, CDCl₃) δ: 6.58(d, 1H), 6.04(d, 1H), 4.47(m, 1H), 4.30(d, 1H),4.17(m, 1H), 3.67(s, 3H), 3.53(m, 2H), 3.2-3.4(t, d, 2H), 2.3(m, 3H),2.2 (t, 1H), 1.83(m, 1H), 1.2-1.8(m, 24H), 1.2(m,3H), 0.9(d, 3H).

Step 2: Monomethylazelayl-N^(g) -nitro-L-arginyl-L-leucinal:

Using the Method E (Example 11), the peptide acetal (from step 1) (250mg) was converted to Compound 26 (0.22 g). ¹ H NMR (300 MHz, DMSO-d₆) δ:9.38(s, 1H), 7.73(d, 1H), 4.30(m, 1H), 4.09(m, 1H),3.57(s, 3H), 3.15(m,2H), 3.01(q, 1H), 2.75(m, 1H), 2.24(m, 7H),1.50(m, 12H), 1.24(b, 14H),0.86(m, 6H)

EXAMPLE 27 Monomethylazelayl-N^(g) -nitro-L-arginyl-L-phenylalaninal##STR34## Step 1. Phenylalaninal diethylacetal.

CBZ-Phe-OH (6.0 g, 2 mmol) was converted to phenylalaninal-diethylacetal following the same procedure used in Leucinal diethylacetal(method F, Example 12).

Step 2: Fmoc-Arg(NO₂)-OH.

A solution of fluorenylmethyloxycarbonyl-N-hydroxysuccinimidyl ester (32mmol) in 60 mL of THF was added to a stirred solution of N^(g)-nitro-L-arginine (35 mmol) and NaHCO₃ (70 mmol) in 70 mL of H₂ O. Themilky solution cleared after 1 h and the solution was acidified withsolid citric acid to pH 2-3 and extracted with 300 mL of EtOAc. Theorganic layer was washed once with water, dried and evaporated to yieldthe compound as white solid (26.8 mmol).

Step 3: Fmoc-Arg(NO₂)-Resin.

A solution of 9-fluorenylmethyloxycarbonyl-N^(g) -nitro-L-arginine (fromstep 2, 26.8 mmol), BOP (30 mmol), HOBt (30 mmol) and NMM (55 mmol) in40 mL of DMF was mixed with 10 g of PAC resin (α-methylphenacyl linkerattached to polystyrene-1% divinylbenzene, substitution 0.97 mmol/g,supplied by Bachem Bioscience, Inc, King of Prussia, Pa.) and stirredfor 4 h. The resin was filtered off, washed with DMF, DCM and MeOH anddried to yield the final product Fmoc-Arg(NO₂)-Resin (11.1 g). Theresin, Fmoc-Arg(NO₂)-Resin (11.1 g) was treated with 100 ml of asolution containing piperidine (30%), DMF (35%) and toluene (35%) andstirred for 2.5 hours. The Fmoc removed resin was filtered andconsecutively washed with DCM/DMF (50:50) and MeOH to yield the product(9.2 g)

Step 4: MeOAz:Arg(NO₂)-Resin.

Monomethyl azelate (20 mmol) was added to a stirred slurry of theArg(NO₂)-Resin (9.2 g), BOP (20 mmol), HOBt (20 mmol) and NMM (to adjustthe pH to 8). After overnight stirring, a mixture ofmonomethylazelate(10 mmol), BOP (10 mmol), HOBt (10 mmol), and NMM (20mmol) was added and stirred for 24 hours. The resin was washed with DMF,DCM and methanol to yield 10.56 g of the resin.

Step 5: Monomethylazelayl-N^(g) -nitro-L-arginine:

The product from step 4(10.56 g) was stirred for 5 h in a solution of100 mL of 67% DCM (30%) TFA and (3%) anisole. The slurry was filteredand the solvent was evaporated and triturated with ether to yield thepeptide (1.11 g, 2.75 mmol).

Step 6: Monomethylazelayl-N^(g) -nitro-L-arginyl-L-phenylalaninaldiethylacetal.

Following Method C (Example 9), methoxyazelaoyl-N^(g) -nitro-L-arginine(1 mmol) was coupled with L-phenylalaninal diethylacetal (1.3 mmol)using BOP (1.3 mmol), HBOt (1.3 mmol) and NMM (to adjust the pH to 8) toyield the title peptide (0.82 mmol).

Step 7: Monomethylazelayl-N^(g) -nitro-L-arginyl-L-phenylalaninal.

Using Method E (Example 11), product (0.82 mmol) from step 6 wasconverted to the title compound (0.81 mmol). ¹ H NMR (300 MHz, CDCl₃) δ9.59(s, 1H), 8.36(s, 1H), 7.49(s, 1H), 7.31-7.09 (m, 7H), 6.71 (d, 1H),4.69 (m, 1H), 4.60 (m, 1H), 3,66 (s, 3H), 3.41 (m, 2H), 3.27 (m, 2H),2.31 (t, 2H), 2.2 (t, 2H), 1.80-1.2 (m, 14H)

EXAMPLE 28 Monomethylazelayl-N^(g) -(2,2,5,7,8-pentamethylchroman-6-sulfonyl)-L-arginyl-L-leucinal ##STR35## Step 1:Monomethylazelayl-N^(g)-(2,2,5,7,8-pentamethylchroman-6-sulfonyl)-L-arginyl-L-leucinaldiethylacetal.

Following Method C (Example 9), monomethylazelate (1.42 g, 7.0 mmol) wascoupled with N^(g)-(2,2,5,7,8-pentamethylchroman-6-sulfonyl)-L-arginyl-L-leucinaldiethylacetal (3.06 g, 5.0 mmol, obtained from step 2 in Example 20),using 7.0 mmol each of BOP, HOBt and NMM to obtain the compound (3.43 g)as solid. ¹ H NMR (300 MHz, CDCl₃) δ: 6.58(d, 1H), 6.04(d, 1H), 5.4(br,1H), 5.06(br, 1H), 4.47 (m, 1H), 4.30 (d, 1H), 4.17 (m, 1H), 3.67 (s,3H), 3.53 (m, 4H), 3.23 (d, 1H), 3.19 (t, 2H), 2.30 (m, 2H), 2.2 (tt,2H), 2.0-1.25 (2 br m, 17H), 1.20 (tt, 6H), 0.90 (dd, 6H).

Step 2: Monomethylazelayl-N^(g)-(2,2,5,7,8-pentamethylchroman-6-sulfonyl)-L-arginyl-L-leucinal.

Using Method E (Example 11), the product (0.30 g) from step 1 wasconverted to Compound 28 (0.22 g). ¹ H NMR (300 MHz, DMSO-d₆) δ: 9.38(s, 1H), 8.5 (br, 1H), 4.30 (m, 1H), 4.09 (m, 1H), 3.57 (s, 3H), 3.15(m, 2H), 3.00 (t, 2H), 2.75 (m, 2H), 2.12 (t, 2H), 1.8-1.20 (2 br m,17H), 0.86 (dd, 6H)

EXAMPLE 29 Monomethylazelayl-N^(g)-(2,2,5,7,8-pentamethylchroman-6-sulfonyl)-D-arginyl-L-leucinal##STR36## Step 1: 9-Fluorenylmethoxycarbonyl-N^(g)-(2,2,5,7,8-pentamethylchroman-6-sulfonyl)-D-arginyl-L-leucinaldiethylacetal.

Following Method C (Example 9), 9-Fluorenylmethyloxycarbonyl-N^(g)-(2,2,5,7,8-pentamethylchroman-6-sulfonyl)-D-arginine (3 mmol) wascoupled with leucinal diethylacetal (2.5 mmol) using BOP (3 mmol), HOBt(3 mmol) and NMM (5 mmol) in 5mL of DMF to yield the peptide (1.72 g, 2mmol) as a solid.

Step 2: MeOAz-D-Arg(PMC)-Leucinal diethylacetal.

Using Method C (Example 9), monomethylazelate (1.0 mmol) was coupledwith N^(g)-(2,2,5,7,8-pentamethylchroman-6-sulfonyl)-D-arginyl-L-leucinaldiethylacetdal (0.54 g, 0.88mmol) obtained from title product of Step 1following method B (Example 8), using BOP (1 mmol), HBOt (1 mmol) andNMM (3 mmol) in 5 mL of DMF to yield the product (0.735 g).

Step 3: Monomethylazelayl-N^(g)-(2,2,5,7,8-pentamethylchroman-6-sulfonyl)-D-arginyl-L-leucinal.

Using method E (Example 11), the product (0.1 g) from step 2 wasconverted to Compound 29 and purified by HPLC. ¹ H NMR(300 MHz, CDCl₃)δ: 9.49 (s,1H), 7.74 (t, 1H), 7.43 (d, 1H), 6.43 (d, 1H), 6.34 (s, 2H),4.60 (m, 1H), 4.51 (s, 3H), 4.54 (s, 3H), 4.37 (m, 1H), 3.66 (s, 3H),3.31 (m, 2H), 2.63 (t, 2H), 2.26 (m, 4H), 2.09 (s, 3H), 1.80 (t, 2H),1.57 (m, 7H), 1.26 (m, 16H), 0.90 (dd, 6H)

EXAMPLE 30 Monomethylazelayl-N^(g)-(2,2,5,7,8-pentamethylchroman-6-sulfonyl)-L-arginyl-L-norleucinal##STR37## Step 1: Fmoc-N^(g)-(2,2,5,7,8-pentamethylchroman-6-sulfonyl)-L-arginyl-L-norleucinal.

Fmoc-Arg(PMC)-OH (2 mmol) was coupled with norleucinal diethylacetal (2mmol, obtained from CBZ-Nle-OH following the procedures in thepreparation of Leu-acetal) using BOP (2 mmol), HOBt (2 mmol) and NMM (6mmol) in 6 mL of DMF, according to the Method C (Example 9), to yieldthe peptide (1.37 g, 1.64 mmol).

Step 2: MeOAz-Arg(PMC)-L-norleucinal diethyl azetal.

Following Method C (Example 9), monomethyl azelate (2 mmol) was coupledwith Arg(PMC)-norleucinal diethyl acetal (1.39 mmol, obtained from step1 following Method B, Example 8) using BOP (2 mmol), HOBt (2 mmol) andNMM (6 mmol) and stirred overnight. The next day, 1 mmol each ofmonomethylazelate, BOP, HOBt and NMM were added and stirred for 4 hours.The reaction mixture was worked-up as in Method C, Example 9), to yieldthe peptide (0.37 g, 0.84 mmol)

Step 3: Following Method E (Example 11), the product from step 2 (0.94g, 0.84 mmol) was converted to Compound 30 (0.58 g) ¹ H NMR(300 MHz,CDCl₃) δ: 9.54 (s, 1H), 7.49 (t, 1H), 6.74 (d, 1H), 6.26 (s, 2H), 6.23(d,1H), 4.58 (m, 1H), 4.31 (m, 1H), 3.66 (s, 3H), 3.34 (m, 2H), 2.63 (t,2H), 2.58 (s, 3H), 2.56 (s, 3H), 2.29 (t, 2H), 2.23 (t, 2H), 1.9-1.5 (m,22H), 1.30 (s, 6H), 0.86 (t, 3H)

EXAMPLE 31 Monomethylazelayl-N^(g)-(p-toluenesulfonyl)-L-arginyl-L-leucinal ##STR38## Step 1: Fmoc-N^(g)-(p-toluenesulfonyl)-L-arginyl-L-leucinal diethylacetal.

Using Method C (Example 9), Fmoc-N⁹ -(p-toluenesulfonyl)-L-arginine (5mmol) was coupled with leucinal diethylacetal (5.5 mmol) using HBTU (5.5 mmol), HOBt (5.5 mmol) and NMM (11 mmol) in 15 mL of DMF. The peptidewas isolated as a solid (2.86 g, 3.96 mmol).

Step 2: Monomethylazelayl-N^(g)-(p-toluenesulfonyl)-L-arginyl-L-leucinal diethylacetal.

Following Method C (Example 9), monomethyL azelate (6 mmol) was coupledwith N^(g) -(p-toluenesulfonyl)-L-arginyl-L-leucinal diethylacetal (2.7g, 4.7 mmol, obtained from Step 1 using Method B, Example 8), using1-benzotriazol-1-yl-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU) (6 mmol), HOBt (6 mmol) and NMM (12 mmol) in 15 mL of DMF andstirring overnight. The reaction yield was boosted by adding monomethylazelate (3 mmol) and diphenylphosphoryl azide (3 mmol) and stirred for 4hours to yield the peptide as solid (1.92 g).

Step 3: Monomethylazelayl-N^(g)-(p-toluenesulfonyl)-L-arginyl-L-leucinal

According to Method E (Example 11), the product from step 2 (1.92 g, 2.8mmol) was converted to Compound 31 (1.64 g) ¹ H NMR (300 MHz, DMSO-d6)δ: 9.37 (s, 1H), 8.97 (d, 1H), 8.37 (d, 1H), 7.66 (d, 2H), 7.37 (m, 1H),7.29 (d, 2H), 7.03 (s, 1H), 6.63 (s, 1H), 4.09 (m, 1H), 3.57 (s, 3H),3.44 (m, 1H), 3.04 (m, 2H), 2.26 (s, 3H), 2.33 (t, 2H), 2.13 (t, 2H),2.80-1.09 (m, 7H), 0.86 (dd, 6H).

EXAMPLE 32 Monomethylazelayl-N^(g)-(4-methoxy,2,3,6-trimethyl-1-sulfonyl)-L-arginyl-L-leucinalsemicarbazone ##STR39##

See Basak, A. et al., Int. J. Peptide Protein Res. 36 7-17 (1990). Amixture of MeOAz-Arg(MTR)-Leu-H (Example 34) (67 mg, 0.10 mmol),semicarbazide hydrochloride (11 mg, 0.1 mmol) and sodium acetate (9 mg,0.11 mmol) in 3 mL of 90% EtOH was heated to 70° C. for 18 h. Thereaction mixture was concentrated to afford a light yellow solid(Compound 32) which was subsequently purified by HPLC as in Table 6. ¹ HNMR (300 MHz, CDCl₃) δ: 7.91 (t, 1H), 7.09 (d, 1H), 6.68 (s, 1H),6.22(s, 1H), 4.37-4.46 (m, 1H), 4.2-4.3 (m, 1H), 4.04 (d, 1H), 3.7 (s, 3H),3.58 (s, 3H), 2.6 (s, 3H), 2.27 (dd, 2H), 2.05, 2.12 (s, 3H),1.2-1.64(m, 12H), 0.85(t, 6H)

EXAMPLE 33 Monomethylazelayl-N^(g)-(4-methoxy,2,3,6-trimethyl-1-sulfonyl)-L-arginyl-L-leucinalthiosemicarbazone) ##STR40##

Following the same steps in Example 32, Compound 33 was made from theCompound of Example 34 (51 mg, 0.07 mmol) and thiosemicarbazide (7 mg,0.07 mmol) in 2 mL of 90% EtOH.

EXAMPLE 34 Monomethylazelayl-N^(g)-(4-methoxy,2,3,6-trimethylbenzene-1-sulfonyl)-L-arginyl-L-leucinal##STR41## Step 1: Following method C (Example 9),9-fluorenyl-methyloxycarbonyl-N^(g)(4-methoxy-2,3,6-trimethyl-benzene-1-sulfonyl)-L-arginyl-L-leucinaldiethyl acetal was prepared from 9-fluorenylmethyloxycarbonyl-N^(g)-(4-methoxy-2,3,6-trimethylbenzene-1-sulfonyl)-L-arginine and L-leucinaldiethyl acetal.

Step 2: Monomethylazelayl-N^(g)-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginyl-L-leucinal.

Following Method C (Example 9), monomethylazelate (6 mmol) was coupledwith N^(g)-(4-methoxy-2,5,6-trimethylbenzene-1-sulfonyl)-L-arginyl-L-leucinaldiethylacetal (5 mmol, obtained from step 1 using Method B, Example 8),and the peptide was isolated as an amorphous solid (3.3 g).

Step 3: Methoxyazelayl-N^(g)-(4-methoxy-2,3,6-trimethylbenzene-1-sulfonyl)-L-arginyl-L-leucinal.

The product from step 2 (0.5 g) was converted to Compound 34 (0.36 g)according to method E, Example 11. ¹ H NMR (300 MHz, CDCl₂) δ: 9.54 (s,1H), 7.51 (s, 1H), 6.74(d, 1H), 6.57 (s, H), 6.40(s, 2H), 6.31 (s, 1H),4.66 (m, 1H),4.41 (m, 1H), 3.87 (s, 3H), 3.70 (s, 3H), 3.33(m, 2H), 2.73(s, 3H), 2.66 (s, 3H), 2.33 (t, 2H), 2.26 (t, 2H), 2.17 (s, 3H),2.00-1.26 (m, 17H), 0.96 (dd, 6H).

EXAMPLE 35 Methoxyazelayl-N^(g)-(2,4,6-trimethylbenzene-1-sulfonyl)-L-arginyl-L-leucinal ##STR42## Step1: Fmoc-N^(g) -(2,4,6-trimethylbenzene-1-sulfonyl)-L-arginyl-L-leucinaldiethylacetal.

A solution of 4M HCl in dioxane (10 mL) was added to a solution ofBoc-Arg(MTS)-OH (6 mmol) in 10 ml, of dioxane. After 30 minutes, thesolvent was removed and ether was added, the precipitate was collectedand dried (3.21 g, 9 mmol). The Arg-MTS-OH hydrochloride was convertedto Fmoc-Arg(MTS)-OH following the procedure as described for thepreparation of the tosyl derivative (Example 31) to yield the titlecompound as a white solid (2.09 g).

Step 2: Fmoc-Arg(MTS)-Leu-acetal.

Following Method C (Example 9), Fmoc-Arg(MTS)-OH (3.361 mmol) wascoupled with leucinal diethylacetal (4 mmol) using HBTU (4 mmol), HOBt(4 mmol) and NMM (10(mmol) in 15 mL of DMF to yield the title peptide(1.05 g)

Step 3: MeOAz-Arg(MTS)-Leu-acetal.

Using Method C, monomethyl azelate (1.2 mmol) was coupled with Arg(MTS)-Leu-acetal (0.85 mmol, obtained from step 2 following Method B,Example 8) using BOP (1.2 mmol), HOBt (1.2 mmol) and NMM (3.6 mmol) in 3mL of DMF and stirred overnight to give the title peptide as asemi-solid (0.59 g).

Step 4: Methoxyazelaoyl-N^(g)-(2,4,6-trimethylbenzene-1-sulfonyl)-L-arginyl-L-leucinal.

According to Method E (Example 11), the product (0.59 g) of step 3 wasconverted to Compound 35 (0.54 g) and purified by HPLC. ¹ H NMR (300MHz, CDCl3) δ: 9.49(s, 1H), 7.53 (s, 1H), 6.90 (s, 2H), 6.81 (d, 1H),6.40 (bs, 3H), 4.61 (m, 1H), 4.38 (m, 1H), 3.67 (s, 3H), 2.67 (s, 6H),2.29 (t, 2H), 2.20 (t, 2H), 2.0-1.20 (m, 19H), 0.91 (dd, 6H)

EXAMPLE 36 6-Cyano-hexane-1-sulfonyl-N^(g)-(2,2,5,7,8-pentamethylchroman-6-sulfonyl)-L-arginyl-L-leucinal##STR43## Step 1: 6-Cyano-hexane-1-sulfonyl-N^(g)-(2,2,5,7,8-pentamethylchroman-6-sulfonyl)-L-arginyl-L-leucinaldiethylacetal.

6-cyano-hexane-1-sulfonyl chloride (0.19g, 0.89 mmol) from step 14 inMethod G (Example 13) was added to a solution of the product (0.55 g,0.899 mmol) from step 2 in Example 20, in 1 mL of DMF and the pH of thesolution was adjusted to 8 using NMM. After 4 hours of stirring, thereaction mixture was worked up as described in Method A (Example 7), toobtain the title compound (0.466 g).

Step 2: 6-Cyano-hexane-1-sulfonyl-N^(g)-(2,2,5,7,8-pentamethylchroman-6-sulfonyl)-L-arginyl-L-leucinal.

Using method E (Example 11), the product (0.297 g) from step 1 wasconverted to Compound 36 (0.22 g) and purified by HPLC as described inthe Table 6: ¹ H NMR(300 MHz, DMSO-d₆) δ: 9.37 (s, 1H), 6.83 (b, 1H),6.43 (b, 1H), 3.88 (m, 1H), 3.77 (m, 1H), 3.01 (m, 2H), 2.80 (m, 2H),2.55 (t, 2H), 2.46 (s, 6H), 2.0 (s, 3H), 1.75 (m, 5H), 1.6-1.3 (m, 15H),1.23 (s, 6H), 0.84 (dd, 6H)

EXAMPLE 37 2-Naphthoyl-N^(g) -nitro-L-arginyl-L-leucinal ##STR44## Step1: 2-Naphthoyl-N^(g) -nitro-L-arginyl-L-leucinal diethylacetal.

2-Naphthoylchloride (0.104 g, 0.55 mmol) was added to a solution of theproduct from step 2 in Example 14 in 2 mL of DMF and NMM (0.18 mL) andthe title product was worked up to obtain the title compound as a solid(22 g). ¹ H NMR (300 MHz, CDCl3) δ: 8.92 (b, 1H), 8.0-7.91 (mm, 10H),6.93 (d, 1H), 5.07 (m, 1H), 4.37 (d, 1H ), 4.17 (m, 1H), 3.69 (m, 3H),3.53 (m, 3H), 3.38 (m, 2H) 1.77, 1.58, 1.4 (mm, 5H), 0.83 (dd, 6H).

Step 2: 2-Naphthoyl-N^(g) -nitro-L-arginyl-L-leucinal.

Using Method E (Example 11), the product (0.1 g) from step 1 wasconverted to the Compound 37 (60 mg). ¹ H NMR (300 MHz, DMSO-d₆) δ: 9.43(s, 1H), 8.72 (d, 1H), 8.53 (m, 2H), 8.0 (m, 6H),7.6 (m, 2H),6.20 (m,1H), 4.57 (m, 1H), 4.14 (m, 1H), 3.92 (m, 1H), 3.2 (m, 2H), 3.0 (d, 1H),1.77 (m, 5H), 0.86 (m, 6H).

EXAMPLE 38 CBZ-7-aminoheptanoyl-N^(g) -nitro-L-arginyl-L-leucinal##STR45## Step 1: CBZ-7-aminoheptanoyl-N^(g) -nitro-L-arginyl-L-leucinaldiethyl acetal.

Following Method C (Example 9), CBZ-7-aminoheptanoic acid (0.7 g, 2.5mmol) was coupled with the product of step 2 in Example 14 (0.78 g, 2.0mmol) using BOP (1.1 g, 2.5 mmol), HOBt (0.34 g, 2.5 mmol) and NMM(0.253 mL, 2.5 mmol) to yield the peptide as a semi-solid, which wasused directly in the next step.

Step 2: CBZ-7-aminoheptanoyl-N^(g) -nitro-L-arginyl-L-leucinal.

Following Method E (Example 11), the acetal from step 1 was converted toCompound 38 (0.8 g) after stirring the reaction for 5 h. ¹ H (300 MHz,DMSO-d₆) δ 9.30 (s, 1H), 8.47 (s, 1H), 8.46 (s, 1H), 8.29 (d, 1H), 8.11(t, 1H), 7.80 (s, 1H), 7.64 (d, 1H), 7.31 (s, 1H), 7.23 (s, 1H), 4.91(s, 2H), 4.23 (m, 1H), 4.00 (m, 1H), 3.51 (q, 2H), 3.27 (m, 2H), 2.89(q, 2H), 2.11 (t, 2H), 2.03 (t, 2H), 1.7-1.00 (m, 10H), 0.77 (dd, 6H)

Examples 39-42 describe the syntheses of the MCP inhibitors listed inTable 7.

                  TABLE 7                                                         ______________________________________                                                        % Inhibition                                                  Example No.     (1 μM) IC.sub.50 nM                                        ______________________________________                                        39 (Isomer a)   31        >1000                                               39 (Isomer b)   21        >1000                                               40              100       6                                                   41 (Isomer a)   99        22                                                  41 (Isomer b)   98        13                                                  42              15        >1000                                               ______________________________________                                    

EXAMPLE 39 10-Cyano-2-cyclopentyl-decanoyl-N^(g)-nitro-L-arginyl-L-leucine chloromethylketone ##STR46##

In this and in the following three examples, the inhibitors of theinvention are prepared by Coupling Procedure II. In each case,10-cyano-2-cyclopentyl-decanoyl-N^(g) -nitro-L-arginine prepared asherein described is coupled to an enzyme-reactive amino acid derivativeto produce the inhibitor. These inhibitors are obtained as a mixture oftwo or more diastereoisomers which in some cases may be separable byHPLC.

A) 10-Cyano-2-cyclopentyl-decanoyl-N^(g) -nitro-L-arginine methyl ester

Following the procedure of Method C (Example 9),10-cyano-2-cyclopentyl-decanoic acid (2.4 g; 11 mmol) from Example 13Step 5, in 26 mL of DMF was stirred with N^(g) -nitro-L-arginine methylester dihydrochloride (2.86 g, 11 mmol), BOP (6.6 g, 15 mmol), HOBt(1.62 g, 12 mmol) and 3.6 mL of NMM (33 mmol) to yield 4.8 g of themethyl ester as a foamy solid. ¹ H NMR: (300 MHz, CDCl₃) δ 8.75 (bs,1H), 7.81 (bs, 2H), 6.42 (d, 1H), 4.67 (t, 1H), 3.82 (s, 3H), 3.75 (m,1H), 3.32 (m, 1H), 2.15 (t, 2H), 2.05-1.12 (m, 28H)

B) 10-Cyano-2-cyclopentyl-decanoyl-N^(g) -nitro-L-arginine

A solution of 5.5 g of the methyl ester from part "A" above in 30 mL ofmethanol was treated with 24 mL of 1.00N aqueous sodium hydroxide. After2 hours, 100 mL of 2% aqueous sodium bicarbonate solution was added, andthe resulting solution was extracted with 100 mL of ether. The aqueouslayer was separated and acidified with 3% aqueous citric acid andextracted with 250 mL of ethyl acetate. The resulting organic layer wasseparated, dried over anhydrous MgSO₄ and evaporated to yield acolorless gum which on trituration with petroleum ether solidified to afine white powder weighing 4.4 g. ¹ H-NMR (300 MHz, CDCl₃) δ 10.6 (bs,1H), 8.75 (bs, 1H), 7.82 (bs, 2H), 6.41 (d, 1H), 4.67 (t, 1H), 3.71 (m,1H), 3.32 (m, 1H), 2.15 (t, 2H), 2.04-1.12 (m, 30H).

C) 10-Cyano-2-cyclopentyl-decanoyl-N^(g) -nitro-L-arginyl-L-leucinechloromethyl ketone.

A mixture of 467 mg (1.0 mmol) of the product from part "B" above and270 mg (1.0 mmol) of leucine chloromethyl ketone hydrochloride (BachemBiosciences, Inc., King of Prussia, Pennsylvania), 440 mg (1.0 mmol) ofBOP and 135 mg (1 mmol) of HOBt in 4.0 mL of DMF was treated with 0.33mL (3 mmol) of NMM. After 4 hours. the mixture was diluted with 75 mL ofethyl acetate, washed with 2% aqueous NaHCO₃, water, 3% aqueous citricacid and finally with water. The organic layer was separated and dried(MgSO₄) and finally evaporated to give a pale yellow, viscous oil. Thiscompound was purified by flash chromatography through a 9×1/2 inchcolumn of silica gel 60-H using ethyl acetate for elution. The resultingsolution was evaporated to give a colorless gum which solidified onstanding in 1:1 ethyl acetate/ether to give 198 mg of colorless solidchloromethyl ketone. HPLC indicated the presence of two diastereoisomerswhich were separated by preparative RP-HPLC. In a water-acetonitrilesolvent gradient (30-80% of acetonitrile in 40 min.) the peaks at 22.58min.(diastereoisomer a) and 23.7 min. (diastereoisomer b) were isolated.

Diastereoisomer a: ¹ H-NMR (300 MHz, CDCl₃) δ 8.53 (bs, 1H), 7.61 (bs,2H), 7.31 (bs, 1H), 6.69 (d, 1H), 4.73 (m, 1H), 4.65 (m, 1H), 4.28 (q,2H), 3.53 (m, 1H), 3.31 (t, 1H ), 2.32 (t, 2H), 1.90-1.11 (m, 31H), 0.93(q, 6H).

Diastereoisomer b: ¹ H-NMR (300 MHz, CDCl₃) δ 8.53 (bs, 1H), 7.61 (bs,2H), 7.31 (bs, 1H), 6.79 (d, 1H), 4.73 (m, 1H), 4.65 (m, 1H), 4.28 (q,2H), 3.53 (m, 1H), 3.31 (t, 1H), 2.32 (t, 2H), 1.90-1.11 (m, 31H), 0.93(q, 6H).

EXAMPLE 40 10-Cyano-2-cyclopentyl-decanoyl-N^(g)-nitro-L-arginyl-boroleucine pinacol ester ##STR47##

A solution of 467 mg (1.0 ml of 10-cyano-2-cyclopentyl-decanoyl-N^(g)-nitro-L-arginine (Example 39, part "B" above) and 264 mg (1.0 mmol) ofboroleucine pinacol ester hydrochloride prepared by the method ofShenvi, U.S. Pat. No. 4,537,773, 440 mg (1.0 mmol) of BOP and 135 mg(1.0 mmol) of HOBt in 5.0 mL of DMF was treated with 0.33 mL (3 mmol) ofNMM. After 2 hours, the mixture was diluted with 75 mL of ethyl acetateand washed with 2% aqueous NaHCO₃ and water, and the organic layer wasseparated, dried (MgSO₄) and evaporated to yield 410 mg of a pale brownpowder. This solid was washed with chloroform to give 290 mg of productas an off-white solid which exhibited a single peak in the HPLC. ¹ H-NMR(300 MHz, CDCl₃) δ 8.53 (bs, 1H), 7.65 (bs, 2H), 6.71 (t, 1H), 4.61 (m,1H), 3.52 (m, 1H), 3.31 (m, 2H), 3.05 (m, 1H), 2.82 (m, 1H), 2.38 (t,2H), 2.06-1.42 (m, 28H), 1.22 (s, 12H), 1.15 (m, 2H), 0.92 (m, 6H).

EXAMPLE 41 10-Cyano-2-Cyclopentyl-decanoyl-N^(g)-nitro-L-arginyl-L-leucine alpha-ketoethylamide ##STR48## A)10-Cyano-2-cyclopentyl-decanoyl-N^(g)-nitro-L-arginyl-(1-ethylaminocarbonyl 1-hydroxy-4-methyl)-2-pentylamide

A solution of 467 mg (1.0 mmol) of 10-cyano-2-cyclopentyl-decanoyl-N^(g)-nitro-L-arginine and 225 mg of 3-amino-2-hydroxy-5-methyl-hexanoic acidN-ethylamide hydrochloride (prepared by the method of Harbeson et al,J.Med.Chem. 37, 2918-29 (1994)) in 5.0 mL of DMF was treated with 440 mg(1 mmol) of BOP, 135 mg (1 mmol) of HOBt and 0.33 mL (3 mmol) of NMM.After stirring for 2 hours., the solution was diluted with 75 mL ofethyl acetate and washed with 2% aqueous NaHCO₃, water, 3% aqueouscitric acid, water and dried (MgSO₄) to yield, after evaporation, 540 mgof the hydroxy compound as an off-white solid. ¹ H-NMR. (300 MHz, CDCl₃)δ 8.53 (bs, 1H), 7.73 (bs, 2H), 7.04 (bm, 1H), 6.83 (t, 1H), 4.52 (m,1H), 4.19 (m, 1H), 4.11 (q, 2H), 3.46 (q, 2H), 3.26 (m, 2H), 2.35 (t,2H), 1.91 (m, 2H), 1.83 (m, 2H), 1.8-1.2 (m, 28H), 1.13 (t, 3H), 0.88(m, 6H).

B) 10-Cyano-2-cyclopentyl-decanoyl-N^(g) -nitro-L-arginyl-L-leucinealpha-ketoethylamide

A solution of 250 mg of the hydroxy compound from part "A" above in 6.0mL of dry dichloromethane was cooled to 0° C. and stirred with 225 mg(ca. 0.5 mmol) of Dess-Martin reagent (D.B.Dess and J.C.Martin, J. Org.Chem. 48, 4156-4158 (1983)).

The reaction was allowed to warm to room temperature and was stirred for2 hours. The cloudy suspension was diluted with 50 mL of ethyl acetateand filtered through a fine sintered-glass filter. The filtrate waswashed with 10% aqueous Na₂ S₂ O₃ and then with saturated NaCl. It wasdried (MgSO₄) and evaporated to give 180 mg of white solid ketoamideproduct. It was purified by preparative RP-HPLC using awater-acetonitrile gradient system (40-70% acetonitrile in 40 min.). Thepeaks at 18.07 min. (diastereoisomer a) and 19.54 min. (diastereoisomerb) were collected.

Diastereoisomer a: ¹ H-NMR: (300 MHz, CDCl₃) δ 8.45 (bs, 1H), 7.58 (bs,2H), 7.04 (bm, 2H), 6.57 (t, 1H), 5.33 (t, 1H), 4.60 (m, 1H), 3.51 (m,1H), 3.33 (m, 3H), 2.35 (t, 2H), 1.91-1.11 (m, 34H), 0.94 (m, 6H).

Diastereoisomer b: ¹ H-NMR (300 MHz, CDCl₃) δ 8.45 (bs, 1H), 7.48 (bs,2H), 7.25 (m, 1H), 7.04 (t, 1H), 6.85 (m, 1H), 6.62 (d, 1H), 5.32 (t,1H), 4.81 (m, 1H), 4.58 (m, 1H), 3.51 (m, 1H), 3.35 (m, 3H), 2.35 (t,2H), 1.95-1.11 (m, 32H), 0.98 (m, 6H).

EXAMPLE 42 10-Cyano-2-cyclopentyl-decanoyl-N^(g)-nitro-L-arginyl-phenylalanine fluoromethylketone ##STR49## A) Synthesisof 1-Nitro-2-phenylethane

To a stirring mixture of trans-β-nitrostyrene (5.25 g, 0.035 mol) andsilica gel (10 g, 230-400 mesh) in chloroform (400 mL) and isopropanol(75 mL) at room temperature, was slowly added sodium borohydride (5.50g, 0.145 mol) over a period of 45 min. The reaction mixture was stirredfor an additional 15 min and then carefully quenched with 10%hydrochloric acid (20 mL). Separated solid wins filtered and washed withchloroform (50 mL). Combined filtrate and washing was washed with water(1×20 mL), brine (1×20 mL) and dried over anhydrous sodium sulfate.Solvent evaporation at reduced pressure gave a crude material which waspurified by flash chromatography (silica gel, 8% ethyl acetate-hexane)to give 2.86 g of 1-nitro-2-phenylethane as a colorless oil (spicyodor); Rf (10% ethyl acetate in hexane): 0.40; ¹ H-NMR (300 MHz, CDCl₃)7.40-7.20 (m, 5H), 4.60 (t, 2H), 3.30 (t, 2H).

B) Synthesis of 1-Fluoro-2-hydroxy-3-nitro-4-phenylbutane

To a cooled (-78° C.) solution of oxalyl chloride (2M) in methylenechloride (11.60 mL, 0.0232mol) was added slowly dimethyl sulfoxide (3.65g, 3.32 mL, 0.0467 mol). The reaction mixture was stirred for 15 min. Asolution of 2-fluoroethanol (1.16 g, 0.0181 mol) in methylene chloride(10 mL) was then slowly introduced into the reaction flask. Afterstirring for another 15 min, the reaction mixture was diluted withanhydrous methylene chloride (180 mL), and triethylamine (9.20 g, 12.63mL, 0.090 mol) was added to it. Stirring was continued for another 2 hby which time the temperature had risen to room temperature. At thistime, a solution of 1-nitro-2-phenylethane (2.74 g, 0.0181 mol) inanhydrous methylene chloride (10 mL) was added to the reaction mixtureand stirring was continued overnight. The mixture was then washed withwater (1×30 mL), 4% hydrochloric acid (3×20 mL), water (1×20 mL),saturated sodium bicarbonate solution (2×20 mL) and brine (1×20 mL).Drying over anhydrous sodium sulfate and solvent evaporation gave acrude material which was purified by flash chromatography (silica gel,25% ethyl acetate-hexane) to give the product as erythro and threoisomers. Combined yield was 3.01 g. A general description of thisprocedure can be found in Imperiali, B., et al., Tetrahedron Lett. 27(2), 135 (1986) and in Revesz, L., et al., Tetrahedron Lett. 35 (52),9693 (1994).

Isomer a was a white solid, mp 71-73° C.; R_(f) (30% ethyl acetate inhexane): 0.46; ¹ H-NMR (300 MHz, CDCl₃) δ 7.40-7.10 (m, 5H), 4.90 (m,1H), 4.60 (m, 1H), 4.50-4.30 (m, 2H), 3.45-3.25 (m, 2H), 2.70 (d, 1H).

Isomer b was a colorless oil; R_(f) (30% ethyl acetate in hexane): 0.42;¹ H-NMR (300 MHz, CDCl₃) δ 7.40-7.15 (m, 5H), 4.90 (m, 1H), 4.65 (m,1H), 4.50 (m, 1H), 4.20 (m, 1H), 3.40-3.30 (m, 2H), 2.90 (d, 1H).

C) Synthesis of 3-Amino-1-fluoro-2-hydroxy-4-phenylbutane

A mixture of the above isomer a (0.48 g, 2.25 mmol), absolute ethanol(20 mL) and Raney-Nickel (catalytic) was hydrogenated (60 psi) in a Parrapparatus for 5 hours. Filtration through a Celite pad and solventevaporation gave 410 mg of amine isomer a. Similar treatment of theabove isomer b (800 mg, 3.75 mmol) gave 510 mg of amine isomer b.

Amine isomer a was a white solid, mp 64-67° C.; ¹ H-NMR (300 MHz, CDCl₃)δ 7.40-7.10 (m, 5H), 4.70 (d, 1H), 4.50 (d, 1H), 3.90-3.70 (m, 1H),3.30-3.10 (m, 1H), 2.95 (dd, 1H), 2.60-2.45 (q, 1H), 2.20-1.70 (broad,3H).

Amine isomer b was a white solid, mp 67-70° C.; ¹ H-NMR (300 MHz, CDCl₃)δ 7.40-7.10 (m, 5H), 4.70 (d, 1H), 4.55 (d, 1H), 3.70-3.50 (m, 1H),3.20-3.00 (m, 1H), 2.95 (dd, 1H), 2.60-2.45 (q, 1H), 2.20-1.65 (broad,3H).

D) 10-Cyano-2-cyclopentyl-decanoyl-N^(g)-nitro-L-arginyl-(4-fluoro-3-hydroxy-1-phenyl)-2-butylamide

A solution of 467 mg (1.0 mmol) of 10-cyano-2-cyclopentyl-decanoyl-N^(g)-nitro-L-arginine and 183 mg (1.0 mmol) of3-amino-1-fluoro-2-hydroxy-4-phenyl-butane in 5.0 mL of DMF was treatedwith 440 mg (1.0 mmol) of BOP, 135 mg (1.0 mmol) of HOBt and 0.33 mL (3mmol) of NMM. After stirring for 2 hours., the solution was diluted with75 mL of ethyl acetate and washed with 2% aqueous NaHCO₃, water, 3%aqueous citric acid, water and dried (MgSO₄) to yield after evaporation480 mg of the hydroxy compound as an off-white solid. ¹ H-NMR (300 MHz,CDCl₃ +d₆ -DMSO) δ 8.15 (bs, 1H), 7.82 (bs, 2H), 7.21 (m, 6H), 5.05 (t,1H), 4.51 (m, 1H), 4.22 (m, 1H), 3.82 (m, 1H), 3.75 (m, 2H), 2.95 (q,2H), 2.35 (t, 2H), 2.04-1.13 (m, 31H).

E) 10-Cyano-2-cyclopentyl-1-decanoyl-N^(g)-nitro-L-arginyl-phenylalanine fluoromethylketone

A solution of 250 mg of the hydroxy compound from part "C" above in 6.0mL of anhydrous dichloromethane was cooled to 0° and stirred with 225 mg(ca. 0.5 mmol) of Dess-Martin reagent. The reaction was allowed to warmto room temperature and was stirred for 2 hours. The cloudy suspensionwas diluted with 50 mL of ethyl acetate and filtered through a finesintered glass filter. The filtrate was washed with 10% aqueous Na₂ S₂O₃ and then with saturated NaCl. It was dried (MgSO₄) and evaporated togive 180 mg of white solid. After HPLC purification, 42 mg of purefluoromethyl ketone product was obtained. ¹ H-NMR: (300 MHz, CDCl₃) δ8.56 (bs, 1H), 7.62 (bs, 2H), 7.42 (t, 1H),7.21 (m, 5H), 6.63 (m, 1H),5.05-4.53(m, 4H), 3.46 (m, 1H), 3.18 (m, 2H), 2.98 (q, 2H), 2.33 (t,2H), 2.04-1.13 (m, 27H).

Each of the published documents referred to in this specification isherein incorporated by reference in its entirety.

Those skilled in the art will appreciate that numerous changes andmodifications may be made to the preferred embodiments of the inventionand that such changes and modifications may be made without departingfrom the spirit of the invention. It is therefore intended that theappended claims cover all equivalent variations as fall within the truespirit and scope of the invention.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 10                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 36 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 #     36           GCCA CCATGGCGAT GAAAGC 36                                  - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 30 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 #           30     ATTA CAGTTTAATG                                            - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 #21                GAAA C                                                     - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 #                24GGAA TGCT                                                  - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 #                24CCGT ACAA                                                  - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 # 20               AGGG                                                       - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 41 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 #   41             GCCA TGGTGGCGGC AAGCTTCGAT C                               - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 # 20               GGGA                                                       - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 41 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 #   41             CACC ATGGCGATGA AAGTGGTGTG C                               - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                #  18              AA                                                         __________________________________________________________________________

What is claimed is:
 1. A composition for the reduction of Cu/Znsuperoxide dismutase-1 enzyme degradation comprising an inhibitor ofmulticatalytic protease and a pharmaceutically acceptable carrier. 2.The composition of claim 1 wherein the inhibitor of multicatalyticprotease is a compound of formula: ##STR50## wherein: R₁ is selectedfrom the group consisting of --C.tbd.N, --C(═O)OR₉, phthalimido,--NH--SO₂ R₉, and --NH--J;R₂ is selected from the group consisting of H,hydroxyl, alkyl having from one to ten carbons, and cycloalkyl havingfrom three to seven carbons; R₃ is selected from the group consisting of--(CH₂)_(m) --NH--C(═N--R₅)--NH₂, --R₆ --NO₂, --R₆ --J, and --R₆ --CN;R₄ is --CH(CH₂ --R₇)--Q; Q is selected from the group consisting of--CH--R₈, --C(═O)CH₃, --C(═O)CH₂ Cl, --C(═O)CH₂ Br, --C(═O)CH₂ F,--C(═O)CHF₂, --C(═O)CF₃, --C(═O)C(═O)R₇, --C(═O)C(═O)NH--R₇, --C(═O)CO₂--R₇, --C(═O)CO₂ H, --B(OH)₂, ##STR51## where p and q, independently,are 2 or 3; W is cycloalkyl; R₅ is selected from the group consisting of--NO₂, --CN, and --J; R₆ is --(CH₂)_(m) --NH--C(═NH)--NH--; R₇ isselected from the group consisting of phenyl, and alkyl having from oneto eight carbons, said alkyl group being optionally substituted with oneor more halogen atoms, aryl, or heteroaryl groups; R₈ is selected fromthe group consisting of ═O, ═N--NHC(═O)--NH₂, ═N--OH, ═N--OCH₃,═N--O--CH₂ --C₆ H₅, ═NNH--C(═S)--NH₂ and ═N--NH--J; R₉ is selected fromthe group consisting of hydrogen, and alkyl having from one to sixcarbons, said alkyl group being optionally substituted with one or morehalogen atoms, aryl or heteroaryl groups; J is a protecting group; n isan integer from 3 to 10; and m is an integer from 2 to
 5. 3. Acomposition for the alleviation of symptoms of a disorder characterizedby a reduction in Cu/Zn superoxide dismutase-1 enzyme activitycomprising an inhibitor of multicatalytic protease and apharmaceutically acceptable carrier.
 4. The composition of claim 3wherein the inhibitor of multicatalytic protease is a compound offormula: ##STR52## wherein: R₁ is selected from the group consisting of--C.tbd.N, --C(═O)OR₉, phthalimido, --NH--SO₂ R₉, and --NH--J;R₂ isselected from the group consisting of H, hydroxyl, alkyl having from oneto ten carbons, and cycloalkyl having from three to seven carbons; R₃ isselected from the group consisting of --(CH₂)_(m) --NH--C(═N--R₅)--NH₂,--R₆ --NO₂, --R₆ --J, and --R₆ --CN; R₄ is --CH(CH₂ --R₇)--Q; Q isselected from the group consisting of --CH--R₈, --C(═O)CH₃, --C(═O)CH₂Cl, --C(═O)CH₂ Br, --C(═O)CH₂ F, --C(═O)CHF₂, --C(═O)CF₃,--C(═O)C(═O)R₇, --C(═O)C(═O)NH--R₇, --C(═O)CO₂ --R₇, --C(═O)CO₂ H,--B(OH)₂, ##STR53## where p and q, independently, are 2 or 3; W iscycloalkyl; R₅ is selected from the group consisting of --NO₂, --CN, and--J; R₆ is --(CH₂)_(m) --NH--C(═NH)--NH--; R₇ is selected from the groupconsisting of phenyl, and alkyl having from one to eight carbons, saidalkyl group being optionally substituted with one or more halogen atoms,aryl, or heteroaryl groups; R₈ is selected from the group consisting of═O, ═N--NHC(═O)--NH₂, ═N--OH, ═N--OCH₃, ═N--O--CH₂ --C₆ H₅,═NNH--C(═S)--NH₂ and ═N--NH--J; R₉ is selected from the group consistingof hydrogen, and alkyl having from one to six carbons, said alkyl groupbeing optionally substituted with one or more halogen atoms, aryl orheteroaryl groups; J is a protecting group; n is an integer from 3 to10; and m is an integer from 2 to
 5. 5. The composition of claim 3wherein the disorder is selected from the group consisting ofamyotrophic lateral sclerosis, Parkinson's disease, Alzheimers'sdisease, Huntington's disease, stroke, trauma, and ischemia.
 6. A methodfor reducing the degradation of Cu/Zn superoxide dismutase-1 enzyme in amammal wherein said degradation is a symptom associated with a diseaseor disorder comprising administering to a patient a therapeuticallyeffective amount of an inhibitor of multicatalytic protease.
 7. Themethod of claim 6 wherein the inhibitor of multicatalytic protease is acompound of formula: ##STR54## wherein: R₁ is selected from the groupconsisting of --C═N, --C(═O)OR₉, phthalimido, --NH--SO₂ R₉, and--NH--J;R₂ is selected from the group consisting of H, hydroxyl, alkylhaving from one to ten carbons, and cycloalkyl having from three toseven carbons; R₃ is selected from the group consisting of --(CH₂)_(m)--NH--C(═N--R₅)--NH₂, --R₆ --NO₂, --R₆ --J, and --R₆ --CN; R₄ is--CH(CH₂ --R₇)--Q; Q is selected from the group consisting of --CH--R₈,--C(═O)CH₃, --C(═O)CH₂ Cl, --C(═O)CH₂ Br, --C(═O)CH₂ F, --C(═O)CHF₂,--C(═O)CF₃, --C(═O)C(═O)R₇, --C(═O)C(═O)NH--R₇, --C(═O)CO₂ --R₇,--C(═O)CO₂ H, --B(OH)₂, ##STR55## where p and q, independently, are 2 or3; W is cycloalkyl; R₅ is selected from the group consisting of --NO₂,--CN, and --J; R₆ is --(CH₂)_(m) --NH--C(═NH)--NH--; R₇ is selected fromthe group consisting of phenyl, and alkyl having from one to eightcarbons, said alkyl group being optionally substituted with one or morehalogen atoms, aryl, or heteroaryl groups; R₈ is selected from the groupconsisting of ═O, ═N--NHC(═O)--NH₂, ═N--OH, ═N--OCH₃, ═N--O--CH₂ --C₆H₅, ═NNH--C(═S)--NH₂ and ═N--NH--J; R₉ is selected from the groupconsisting of hydrogen, and alkyl having from one to six carbons, saidalkyl group being optionally substituted with one or more halogen atoms,aryl or heteroaryl groups; J is a protecting group; n is an integer from3 to 10; and m is an integer from 2 to
 5. 8. A method for alleviating asymptom characteristic of a disorder characterized by a reduction inCu/Zn superoxide dismutase-1 enzyme activity comprising administering toa patient an inhibitor of multicatalytic protease.
 9. The method ofclaim 8 wherein the inhibitor of multicatalytic protease is a compoundof formula: ##STR56## wherein: R₁ is selected from the group consistingof --C.tbd.N, --C(═O)OR₉, phthalimido, --NH--SO₂ R₉, and --NH--J;R₂ isselected from the group consisting of H, hydroxyl, alkyl having from oneto ten carbons, and cycloalkyl having from three to seven carbons; R₃ isselected from the group consisting of --(CH₂)_(m) --NH--C(═N--R₅)--NH₂,--R₆ --NO₂, --R₆ --J, and --R₆ --CN; R₄ is --CH(CH₂ --R₇)--Q; Q isselected from the group consisting of --CH--R₈, --C(═O)CH₃, --C(═O)CH₂Cl, --C(═O)CH₂ Br, --C(═O)CH₂ F, --C(═O)CHF₂, --C(═O)CF₃,--C(═O)C(═O)R₇, --C(═O)C(═O)NH--R₇, --C(═O)CO₂ --R₇, --C(═O)CO₂ H,--B(OH)₂, ##STR57## where p and q, independently, are 2 or 3; W iscycloalkyl; R₅ is selected from the group consisting of --NO₂, --CN, and--J; R₆ is --(CH₂)_(m) --NH--C(═NH)--NH--; R₇ is selected from the groupconsisting of phenyl, and alkyl having from one to eight carbons, saidalkyl group being optionally substituted with one or more halogen atoms,aryl, or heteroaryl groups; R₈ is selected from the group consisting of═O, ═N--NHC(═O)--NH₂, ═N--OH, ═N--OCH₃, ═N--O--CH₂ --C₆ H₅,═NNH--C(═S)--NH₂ and ═N--NH--J; R₉ is selected from the group consistingof hydrogen, and alkyl having from one to six carbons, said alkyl groupbeing optionally substituted with one or more halogen atoms, aryl orheteroaryl groups; J is a protecting group; n is an integer from 3 to10; and m is an integer from 2 to
 5. 10. A composition for inhibitingmulticatalytic protease comprising a pharmaceutically acceptable carrierand a compound of formula: ##STR58## wherein: R₁ is selected from thegroup consisting of --C.tbd.N, --C(═O)OR₉, phthalimido, --NH--SO₂ R₉,and --NH--J;R₂ is selected from the group consisting of H, hydroxyl,alkyl having from one to ten carbons, and cycloalkyl having from threeto seven carbons; R₃ is selected from the group consisting of--(CH₂)_(m) --NH--C(═N--R₅)--NH₂, --R₆ --NO₂, --R₆ --J, and --R₆ --CN;R₄ is --CH(CH₂ --R₇)--Q; Q is selected from the group consisting of--CH--R₈, --C(═O)CH₃, --C(═O)CH₂ Cl, --C(═O)CH₂ Br, --C(═O)CH₂ F,--C(═O)CHF₂, --C(═O)CF₃, --C(═O)C(═O)R₇, ##STR59## --C(═O)C(═O)NH--R₇,--C(═O)CO₂ --R₇, --C(═O)CO₂ H, --B(OH)₂,where p and q, independently,are 2 or 3; W is cycloalkyl; R₅ is selected from the group consisting of--NO₂, --CN, and --J; R₆ is --(CH₂)_(m) --NH--C(═NH)--NH--; R₇ isselected from the group consisting of phenyl, and alkyl having from oneto eight carbons, said alkyl group being optionally substituted with oneor more halogen atoms, aryl, or heteroaryl groups; R₈ is selected fromthe group consisting of ═O, ═N--NHC(═O)--NH₂, ═N--OH, ═N--OCH₃,═N--O--CH₂ --C₆ H₅, ═NNH--C(═S)--NH₂ and ═N--NH--J; R₉ is selected fromthe group consisting of hydrogen, and alkyl having from one to sixcarbons, said alkyl group being optionally substituted with one or morehalogen atoms, aryl or heteroaryl groups; J is a protecting group; n isan integer from 3 to 10; and m is an integer from 2 to
 5. 11. A methodfor inhibiting multicatalytic protease comprising contacting amulticatalytic protease with an inhibitory amount of a compound offormula: ##STR60## wherein: R₁ is selected from the group consisting of--C.tbd.N, --C(═O)OR₉, phthalimido, --NH--SO₂ R₉, and --NH--J;R₂ isselected from the group consisting of H, hydroxyl, alkyl having from oneto ten carbons, and cycloalkyl having from three to seven carbons; R₃ isselected from the group consisting of --(CH₂)_(m) --NH--C(═N--R₅)--NH₂,--R₆ --NO₂, --R₆ --J, and --R₆ --CN; R₄ is --CH(CH₂ --R₇)--Q; Q isselected from the group consisting of --CH--R₈, --C(═O)CH₃, --C(═O)CH₂Cl, --C(═O)CH₂ Br, --C(═O)CH₂ F, --C(═O)CHF₂, --C(═O)CF₃,--C(═O)C(═O)R₇, ##STR61## --C(═O)C(═O)NH--R₇, --C(═O)CO₂ --R₇,--C(═O)CO₂ H, --B(OH)₂,where p and q, independently, are 2 or 3; W iscycloalkyl; R₅ is selected from the group consisting of --NO₂, --CN, and--J; R₆ is --(CH₂)_(m) --NH--C(═NH)--NH--; R₇ is selected from the groupconsisting of phenyl, and alkyl having from one to eight carbons, saidalkyl group being optionally substituted with one or more halogen atoms,aryl, or heteroaryl groups; R₈ is selected from the group consisting of═O, ═N--NHC(═O)--NH₂, ═N--OH, ═N--OCH₃, ═N--O--CH₂ --C₆ H₅,═NNH--C(═S)--NH₂ and ═N--NH--J; R₉ is selected from the group consistingof hydrogen, and alkyl having from one to six carbons, said alkyl groupbeing optionally substituted with one or more halogen atoms, aryl orheteroaryl groups; J is a protecting group; n is an integer from 3 to10; and m is an integer from 2 to 5.